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Elucidating the enzymatic mechanisms of biological processes by in-cell abiogenetic and in vitro biochemical analyses can be alarming because of the brief and ambiguous attributes of acknowledgment intermediates, which ascertain the acknowledgment mechanisms. Although genomics reveals the abiding end articles of reactions basal genome alternation and evolution—the new DNA sequences created—the intermediates themselves are hardly advised directly, abnormally in alive cells. Here, we appraise reactions of DNA adjustment by akin recombination (HR), which can abet genome instability/evolution. We abode the engineering and use of complete proteins to detect, quantify, trap, and map a axial DNA average in HR, the Holliday alliance (HJ): a four-way DNA alliance [Fig. 1, A (iv) and B]. We use these engineered proteins to abode axiological questions in genome adherence and archetype in distinct alive actual cells: What is the primary use of/need for HR and how does a archetypal RecQ ancestors protein advance genome stability?

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(A) Archetype of an HR archetypal that includes HJs (with blush hexagons, RuvC, on them; see argument S1 for HR and this model). Commutual alongside lines, base-paired DNA strands; abject lines, new DNA synthesis. (B) Illustration of RuvC (blue triangles) bounden to HJ, acclimatized with permission from West (32). (C) Design of chromosomal regulatable ruvCDefgfp gene (see additionally fig. S1). PN25tetO, doxycycline-inducible apostle (39); arrows, admonition of transcription. (D) RDG protein is induced with doxycycline (doxy; Western blot). (E) RDG protects HJ DNA (not beeline bifold DNA; fig. S2D) from Eco RI breach in solution. Left: Eco RI armpit in complete anchored HJc DNA, 3 bp from the HJ center. Middle: Representative assimilation of HJc by Eco RI inhibited by prebinding (fig. S2B) of RuvCGFP or RDG. Right: DNA bandage intensities normalized to that of HJc at time 0 of Eco RI appraisal (means ± SEM, three experiments). (F) RDG accumulation causes dominant-negative UV ablaze sensitivity, implying bootless DNA repair. The abstracts betoken that RuvC protein cannot act on HJs trapped by RDG. Built-in ruvC locus, either ruvC or deleted (ΔruvC) (left), and the protein produced from the chromosomal transgene (right): PN25tetO-RDG, PN25tetO-RuvCGFP, or PN25tetO apostle only. (G) In-cell titration of RDG/RuvC ratios shows that RDG charcoal dominant-negative, implying HJ trapping, back RDG levels are bargain to acquiesce abounding RuvC homodimers. RuvC homodimers/RDG homodimers at 2.3 (black line) bent by Western blots. Right: percentages of RuvC homodimers and RuvC/RDG heterodimers accepted at this ratio. RuvC and RDG levels controlled by IPTG-inducible Ptac and doxycycline-inducible PN25tetO, respectively. MW, atomic weight. (H) RDG inhibits HR in a phage P1 transduction appraisal in the attendance (RDG) or absence of built-in RuvC or RecG HJ resolution proteins and in the absence of both (RDG ruvC, RDG recG, and RDG ruvC recG, respectively). RDG archetype induced (green) or repressed (gray). † or †, abundance < 1 × 10−7 colony-forming units (cfu) per particle; *P < 0.05 about to the uninduced control. *P < 0.05 about to the uninduced RDG ascendancy strain. (I) RDG protects HJs from RusA HJ endonuclease in alive cells. RusA produced from an IPTG-inducible plasmid bargain RDG ad-lib HJ foci (foci declared in Fig. 2) back produced afore (right) but not afterwards RDG (left). Larboard bar in anniversary panel, no IPTG induction. *P < 0.05, two-tailed commutual t test.

HR mends damaged DNA (text S1 and Fig. 1A) and in accomplishing so additionally promotes genome alternation that drives blight (1) and change (2), adjustment of bacilli to our accustomed systems (3), and accustomed defenses (4). Best of the cancer-driving roles of HR action in actual beef acclimation (mostly) autogenous DNA accident (5). Although HR frequently occurs amid identical sister chromosomes in actual beef (6), nonidentical akin chromosomes sometimes recombine, which can annual “loss of heterozygosity” of alleles that advance blight back aberrant (7). Additionally in actual cells, HR amid afresh sequences promotes some cancer-driving chromosomal rearrangements, including ample and baby deletions and duplications (copy cardinal alterations) and translocations (5, 8). In addition, animal RAD51, the ortholog of Escherichia coli RecA—the conserved, ubiquitous, axial HR catalyst—is overexpressed in assorted animal tumors and associated with poor cast (9, 10), implicating HR in the aliment or progression of blight (11).

Here, we ascertain the primary uses of HR in distinct alive actual or abundant (nonsexual) cells: the capital DNA accident types that necessitate HR and the capital cellular processes that annual them. We appraise how accepted HR is in actual cells. The accent of HR would be altered whether acclimated already per genome archetype [estimated for E. coli and analogously per abject brace for animal DNA (12–14)] rather than already per hundred replications. We additionally authenticate a genome-scale ascendancy and directionality of double-strand breach (DSB) adjustment in the E. coli chromosome and abode the functions of a archetypal RecQ ancestors protein in distinct alive cells.

Five animal orthologs of E. coli RecQ DNA helicase are genome-stabilizing blight blockage proteins (15) important to animal health, but their absolute functions accept been ambiguous in alive cells. In biochemical experiments, antiseptic RecQ both promotes RecA-mediated fiber exchange, arch to HJs (16), and disentangles archetypal bifold HJs (17). Although RecQ promotes the net accretion of HJs in alive beef (18) and allows the abasement of DNA at adjourned archetype forks (19, 20), whether it acts afore or afterwards HJs in beef is alien (18), as are its capital roles in cells. Of the bristles animal orthologs, WRN (and aggrandize Sgs1) acts nonredundantly to abate HJ levels in beef (21, 22). Sgs1 additionally works redundantly in DSB resection/HJ advance (23–25), and RECQL4 is alive in this role (26). BLM is alive in HJ-level abridgement (15). RECQL5 may act mainly on RNA (27). RECQL1, RECQL5, and RECQL4 additionally anticipate genome alternation and blight in bodies and mice (27–29), but whether via HR, and at what stage(s), is unknown.

We present engineered protein derivatives of E. coli RuvC four-way DNA alliance (HJ)–specific endonuclease (Fig. 1B) (30–32) that allurement HJs, arrest their added allure with both antiseptic proteins in solution, and in cells, characterization and quantify HJs as beaming foci in distinct alive cells, and map sites of HJs in genomes via chromatin immunoprecipitation sequencing (ChIP-seq). HJs can anatomy both as intermediates in HR, and HR apart back archetype forks arrest and “regress” (33, 34). We analyze HR-generated HJs from non-HR–HJs in E. coli via the requirements of HR-HJ accretion for specific HR proteins not adapted for angle corruption in alive E. coli (33), and as predicted by the biochemistry of the RecA and RecA-loader proteins (35). We ascertain the capital sources and ante of accretion of HR-HJs in abundant E. coli and that the genomic aisle of HJs in DSB adjustment actualization chromosomal directionality. We additionally ascertain a atypical “junction-guardian” role of RecQ, both announcement the accretion of HR-HJs and preventing the accretion of non-HR–HJs. By mining animal blight RNA data, we accuse the RecQ orthologs BLM and RECQL4 in agnate roles in abounding animal cancers.

We engineered endonuclease-defective, beaming protein fusions of four-way DNA junction–specific RuvC by substituting bases encoding catalytic amino acids (described in fig. S1 and argument S1 for four-way alliance specificity) (30–32). We additionally congenital an identical C-terminal blooming beaming protein (GFP) admixture to anatomic RuvC. RuvCDefGFP (RDG), RuvCDefmCherry (RDM), and RuvCGFP are encoded (separately) as doxycycline-inducible transgenes in a nongenic armpit amid the mntH and nupC genes in the E. coli chromosome (Fig. 1, C and D), in beef that additionally accept either the wild-type (WT) or deleted ruvC gene at the built-in locus, as indicated. We antiseptic RDG and RuvCGFP proteins and accepted that both bind archetypal HJs in band-aid (fig. S2, A and B). RuvCGFP cleaves a archetypal HJ, allegedly audacious by the GFP tag (fig. S2C). RDG does not carve the archetypal HJ (fig. S2C), advertence that, as designed, RDG binds but does not carve HJs in solution.

Two assays actualization that RDG inhibits the activities of added proteins at HJs, that is, “traps” HJs in solution. First, prebinding of either RDG or RuvCGFP to a archetypal HJ with an Eco RI acceptance arrangement abreast the alliance (fig. S2B) slowed breach by Eco RI endonuclease of HJ DNA (Fig. 1E) but not beeline DNA (fig. S2D), advertence that both arrest Eco RI accurately at a HJ. Second, we performed antagonism assays amid RDG and Flp high-affinity site-specific recombinase/HJ resolvase (36), appear to accept almost agnate HJ affection to RuvC (32). Added than bisected (55 ± 2%; beggarly ± SEM, three experiments) of the RDG apprenticed to a archetypal HJ absolute the Flp acceptance arrangement resisted displacement by Flp (fig. S2, E to G). We achieve that RDG has HJ-trap action in band-aid with affection agnate to Flp.

Three assays authenticate that RDG inhibits HJ processing by added proteins in E. coli. First, RDG protects adjoin RuvC, causing a “dominant-negative” acuteness to ultraviolet (UV) light. ΔruvC beef are UV-sensitive (Fig. 1F, ΔruvC compared with ruvC ) (32) and became aggressive with RuvCGFP accumulation from the chromosomal transgene (Fig. 1, C and F, ΔruvC PN25tetO-RuvCGFP), adventurous that RuvCGFP commissioned for RuvC. Thus, RuvCGFP was anatomic in beef as it was in band-aid (fig. S2C). By contrast, accumulation of RDG acquired a UV acuteness agnate to that of ΔruvC beef alike in beef that additionally backpack the built-in WT ruvC gene (Fig. 1F, ruvC PN25tetO-RDG; fig. S3, added controls), implying that RDG blocked the action of RuvC on HJs. Because RuvC functions as a dimer (Fig. 1B) (32), we complete regulatable chromosomal cassettes for both RuvC and RDG to alter their ratios. We activate that RDG prevents RuvC action, causing a dominant-negative UV sensitivity, alike back the molar ratios were adapted to aftermath a predicted 36% active-form RuvC homodimers (Fig. 1G, atramentous line) and 16% RDG homodimers (48% heterodimers), adventurous aloof affiliation of the RuvC and RDG monomer subunits. Because the Western blots admeasurement denatured proteins (Fig. 1G and fig. S4B), this is predicted rather than abstinent directly. Back RuvC homodimers were predicted to outnumber RDG at 50% RuvC to 8% RDG (41% heterodimers), beef became aggressive to UV [fig. S4, A and B; 10−1 mM isopropyl-β-d-thiogalactopyranoside (IPTG)]. The abstracts betoken that RDG accessories HJs in alive E. coli, inhibiting RuvC action, alike back RDG homodimers are abandoned about bisected as abundant as anatomic RuvC homodimers (Fig. 1G), but not back RuvC homodimers beat RDG homodimers by a agency of ≥6 (fig. S4, A and B). In abstracts abundant below, we produced RDG homodimers in ≥50-fold balance of built-in RuvC homodimers (fig. S4C) to abduction best or all HJs.

Second, RDG additionally inhibited recombination of beeline DNA with the E. coli chromosome [transductional HR; per Magner et al. (18)], implying inhibition of the pro-HR activities of HJ-processing proteins, including RuvC and RecG. In transductional HR, RuvC and RecG partially acting for anniversary added such that beef deleted for ruvC or recG are somewhat HR-deficient, and ruvC recG bifold mutants are far added HR-deficient (Fig. 1H, gray bars; RDG not induced, analyze RDG with its ΔruvC and ΔrecG derivatives and both with ΔruvC ΔrecG) (32). We activate that consecration of RDG (Fig. 1H, blooming symbols) bargain transductional HR by 6.6 ± 0.01 times in the attendance of autogenous RuvC and RecG proteins (Fig. 1H, RDG blooming bar, induced, compared with RDG gray bar, not induced), behindhand of built-in RuvC (Fig. 1H, RDG ruvC blooming bar), and somewhat added in ΔrecG or ΔruvC ΔrecG beef (Fig. 1H, green). The abstracts betoken that RDG blocks both RuvC and allotment of RecG HR-promoting action on DNA in alive cells.

Last, RDG blocks the action of the RusA HJ and three-way alliance endonuclease [reviewed by Mahdi et al. (37)] in alive E. coli. In the afterward section, we actualization that RDG beaming foci accord with HJs. Here, we acclimated timed accumulation abstracts with regulatable RusA and RDG to actualization that the numbers of ad-lib foci of RDG are bargain if RusA is produced afore RDG but are not bargain if RDG is produced aboriginal (Fig. 1I). The abstracts betoken that back RusA is produced first, it reduces cellular levels of ad-lib HJs (Fig. 1I, right), the butt of which become RDG foci back RDG is produced. Further, back RDG was produced first, RusA had no cogent aftereffect on levels of ad-lib RDG foci (Fig. 1I, left). The abstracts betoken that RDG blocks the nuclease action of RusA on HJs, credible as foci in alive E. coli cells.

We actualization that RDG forms beaming foci that are activated with HJs from HR-DSB adjustment in E. coli beef (for example, Fig. 1A, HR adjustment model), as follows. We induced low levels of chromosomally encoded I–Sce I double-strand endonuclease (38) in proliferating E. coli with the I–Sce I breach armpit either abreast to or far from the archetype abettor (ori) (I-sites, red arrows, DSB; Fig. 2A) to actualize added or beneath reparable DSBs, respectively, in the added and beneath copies of those two chromosomal regions acquired by archetype (diagrammed in Fig. 2A). We accurate the cogwheel numbers of DSBs per corpuscle as beaming foci of GamGFP, a DSB-specific allurement protein (Fig. 2B), per Shee et al. (39).

(A to E) Alternation of RDG foci with HR-DSB repair–generated HJs per alive cell. (A) Strategy for E. coli chromosome breach with chromosomally encoded I–Sce I endonuclease at engineered cut sites (red arrows, DSB). Because of DNA replication, proliferating beef accept added copies of oriC-proximal than oriC-distal DNA and appropriately will accept added DSBs (and HR repair) per corpuscle back broken by I–Sce I ori-proximally than ori-distally. (B) DSBs quantified as GamGFP focus, per Shee et al. (39), actualization that best of the beef with the oriC-proximal cut armpit accept >1 GamGFP/DSB focus, and those with the ori-distal cut armpit accept mostly 1 focus per cell, as advanced declared by Shee et al. (39). (C) Representative images of RDG foci afterwards I–Sce I breach (top row) or ad-lib foci (bottom row). (D) I–Sce I–induced RDG foci are absolutely activated with numbers of DSBs (and HJs) [quantification of images as in (B)], analogously to GamGFP foci in both oriC-proximal and oriC-distal sites. RDG foci access with DSBs (P = 0.0001 for anniversary locus, two-tailed unpaired t test), and added beef with >1 focus with ori-proximal than ori-distal breach (P = 0.0001, two-tailed unpaired t test). (E) HR protein assurance of I–Sce I/DSB–induced RDG foci. I–Sce I/DSB–induced RDG foci are bargain by a cogent 26.1 ± 0.1 times in recA and 4.3 ± 0.1 (means ± SEM) times in recB absent mutants advertence RecAB dependence, acknowledging their estimation as HR-dependent foci formed during DSB repair. The assurance of RDG focus accretion on RuvB supports their accretion at four-way junctions. The adeptness of ruvA implies that RDG binds anon to DNA four-way junctions, as credible biochemically (44). **P < 0.01, ***P < 0.001, about to DSB control, two-tailed unpaired t test. (F) RDG foci are absolutely activated with dosage of DSB-inducing γ radiation (R2 = 0.99; P < 0.001, Pearson’s alternation analysis). (G) RDG ChIP-seq shows that RDG localized DNA abreast a reparable I–Sce I–induced DSB (vertical red line) in the E. coli chromosome. I-site L, I–Sce I endonuclease armpit L. The ample peaks abandon in DSB− beef (cells accustomed the cut armpit but no I–Sce I enzyme). RDG ChIP-seq reads are normalized to the absolute reads in anniversary sample and added normalized to the ascribe genomic DNA. (H) RDG ChIP-seq shows accessory at a altered chromosomal I–Sce I breach site: I-site J, almost centermost amid the archetype abettor and abuttals in the E. coli “right” replichore. The I–Sce I–induced RDG ChIP-seq aiguille is RecA- and RecB-dependent (indicating HR at a DSB), RecF-independent (indicating accretion not at single-strand gaps), and RuvA-independent (supporting absolute bounden of RDG to four-way junctions, as for antiseptic RuvC) (44). Figure S6B shows the DSB− (enzyme no cut site) controls. WGS, whole-genome sequencing.

We acquisition that DSB adjustment induces RDG foci; we activate 11 ± 3 and 9 ± 3 times added beef with RDG foci (means ± SEM) with I–Sce I breach than in uncleaved ascendancy beef (cut site, no enzyme; Fig. 2, C and D; ethics are for ori-proximal and ori-distal cleavage, respectively). Because of the added DNA copies abreast the ori acquired by archetype (Fig. 2A), both added DSBs (Fig. 2B) and added opportunities for adjustment with a abeyant uncleaved sister chromosome are accepted for ori-proximal than ori-distal DSBs (Fig. 2A). We activate that ori-proximal breach of the chromosome produced 10 ± 3 times added beef with >1 RDG focus than did ori-distal breach (Fig. 2D). These abstracts associate the cardinal of RDG foci with DSBs accepted to be adeptness HR adjustment via HJs. The abstracts additionally announce that assorted contest can be visualized as >1 focus per cell—the foci do not adhere into a distinct spot.

Further, RecA and RecB proteins, which are adapted for HJ accretion during HR-DSB adjustment (Fig. 1A) (40, 41), were adapted for I–Sce I–induced RDG focus accretion (Fig. 2E). This supports the estimation that DSB-induced RDG foci announce HR-HJs. RecF endless RecA at non–DSB-instigated HR contest (40–43) and is not adapted for I–Sce I–induced RDG foci, as accepted (Fig. 2E).

RDG foci are additionally activated with numbers of HR-reparable DSBs produced by gamma application (Fig. 2F and argument S2). In argument S2, these and added abstracts are acclimated to appraisal an adeptness of RDG apprehension of HJs of about 50%.

Four added curve of affirmation abutment the cessation that the RDG foci represent HJs. (i) I–Sce I–induced (Fig. 2E) and ad-lib (Fig. 3A) RDG focus accretion requires RuvB, which stabilizes antiseptic RuvC bounden to HJs in band-aid (32), and does not crave RuvA, which is not adapted for RuvC HJ bounden biochemically (44). Antiseptic RuvC can recruit RuvB to archetypal HJs in the absence of RuvA protein in band-aid (45). Because of the specificity of RuvC for HJs (text S1) (30) and the adeptness of RuvB to balance RuvC at HJs (45), our abstracts betoken that in alive cells, RuvC/RDG can additionally bind HJs afterwards RuvA and can recruit and be counterbalanced by RuvB, implying that RDG foci announce HJs.

(A) Non-DSB accident provokes best ad-lib HJs in vegetative, growing E. coli: RecB adeptness indicates that best ad-lib DNA accident repaired is not DSBs. RecF assurance implies ssDNA gaps (42, 43). RuvB dependence: four-way junction–specific. recG aberrant has added ad-lib RDG foci, advertence that RecG reduces the steady-state akin of ad-lib HJs, as predicted by Whitby et al. (91). (B) RecA assurance of the ad-lib HR-HJ foci in the assorted mutants. (C and D) DSB adeptness of best ad-lib HJ foci. Gam, DSB-specific DNA end–binding protein (92, 93) blocks I–Sce I–induced HJ foci (C), but does not block best ad-lib HJ/HR adjustment foci (D). n.s., not cogent (control, fig. S13). (E) Archetype assurance of best ad-lib RDG HJ foci. Ad-lib HR-HJ foci are bargain in dnaATS beef about to WT at nonpermissive temperature (42°C), at which archetype admission is blocked. P = 0.03, two-tailed unpaired t test. (F to I) Microfluidic time-lapse imaging shows the birth, bearing dependence, and alternation of ad-lib RDG HJ foci. (F) Representative images of microcolony with RDG foci (blue arrows) in WT strain. (G) Altitude of fates of ad-lib RDG HR-HJ foci and beef that access them; best ad-lib RDG foci abide over hours afterwards formation; best beef with RDG foci cease to divide, advertence that the bearing of anniversary ad-lib focus letters on a new HJ event. (H) Altitude of bearing assurance of the bearing of ad-lib RDG foci. Accelerated advance in glucose was followed by abrasion beef in the aforementioned average defective glucose to apathetic and arrest corpuscle accommodation in the WT strain. Dejected circles, cardinal of corpuscle divisions; blooming circles, accumulative cardinal of ad-lib foci that appear in anniversary microfluidic microcolony. (I) Ante of ad-lib RDG focus accretion in accelerated and anchored advance phases credible in (F).

Next, (ii) accumulation of HJ endonuclease RusA afore RDG bargain the cardinal of ad-lib RDG foci, implying that the foci announce HJs (Fig. 1I). (iii) The mCherry-tagged RDM forms ad-lib foci that colocalize with foci of a partial-function aberrant RecA-GFP admixture protein (fig. S5) (46), but not with GFP alone, acceding RDM foci in the cellular about of DNA accident or adjustment (fig. S5). Foci of proteins apprenticed to authentic DNA sites are calmly credible at sites afar by 55 kb (39) [13 kb for Wang and Sherratt (47)], and are afar at ≥80 kb (39, 47), such that the colocalization actuality puts RDG almost abreast RecA-bound DNA. (iv) Application ChIP-seq in the afterward section, we authenticate that RDG binds sites of HR-DSB repair. We achieve that RDG foci announce four-way DNA junctions in distinct alive cells.

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We mapped the genomic adjustment mural of RDG at sites of DSB adjustment induced by I–Sce I breach by ChIP-seq application an antibacterial adjoin RuvC. Figure 2G shows cogent accessory of DNA sequences abreast I-site L (red line), abreast the chromosomal archetype origin, oriC (black line), and afterwards of I-site L in the unidirectional archetype aisle (fig. S6, added controls). RDG accessory extends 63 to 73 kb ori-proximally and 169 to 173 kb ori-distally from the cut armpit (changepoint analysis; two absolute experiments). Surprisingly, a abate accessory occurred abreast the archetype abuttals (Fig. 2G), possibly from break-induced adjustment archetype HJs that abide with the archetype angle to the abuttals (see Discussion). Both areas of accomplished reads are DSB-dependent and are not empiric in beef afterwards I–Sce I breach (DSB−, Fig. 2G). Therefore, the accessory reflects bounden of RDG to DNA associated with DSB repair.

In Fig. 2H, with breach about centermost amid the archetype ori and terminus, reads were afresh accomplished at the I-site and preferentially afterwards in the archetype aisle (143 to 145 kb ori-proximally and 207 to 207 kb ori-distally, changepoint analysis; two experiments). The RDG-DNA bounden adapted the strand-exchange (HJ-producing) protein RecA and its DSB-specific loader (48) RecB (Fig. 2H), advertence HR-DSB adjustment specificity, and was absolute of RecF [RecA loader at single-stranded DNA (ssDNA) gaps (42, 43)] and RuvA (Fig. 2H), which is not adapted for RuvC bounden biochemically (44). We achieve that the ChIP-seq maps of RDG bounden actualization the genomic locations of HJs formed by HR-DSB repair. These aboriginal glimpses of the genomic aisle of HJs during DSB adjustment authenticate a directionality of DSB adjustment forth the chromosome not empiric previously, with added HJs ori-distally than ori-proximally (see Discussion).

With no DNA accident induced, RDG foci appeared spontaneously (figs. S7 and S8, A to C), most, we show, as a aftereffect of ad-lib DNA accident and HR repair. Actualization of best ad-lib RDG foci adapted the RAD51-orthologous strand-exchange HR adjustment protein RecA and its loader at ssDNA gaps (42, 43), RecF (Fig. 3, A and B, and fig. S8A), which is akin with RAD52, the animal RAD51 loading alertness protein (49). Antiseptic RecA aids accretion of regressed forks (RFs) in band-aid (50), but RecA is not adapted for the accretion of, or RuvABC action on, RFs in alive E. coli (33) and is adapted for HR adjustment (40, 41). About 75% of ad-lib RDG foci adapted RecA, RecF, and RuvB (Fig. 3A), acknowledging their abettor as ad-lib HR adjustment events. The RuvB assurance supports focus accident at HJs. The altered numbers of foci in these strains do not aftereffect from altered advance rates/numbers of archetype forks, as credible in fig. S9. Mutants that abridgement RecA and additionally RuvB or RecF actualization no added abatement in ad-lib RDG foci than recA distinct mutants, advertence that those proteins advance ad-lib RDG foci RecA dependently (Fig. 3B). The abettor of the 25% RecA-independent ad-lib foci will be addressed in a abstracted study.

Surprisingly, best HR protein–dependent RDG/HJ foci do not aftereffect from DSB repair, as credible in two ways. E. coli HR-DSB adjustment requires RecBC [RecA loader at DSBs (51)], akin with animal BRCA2, which endless RAD51 at DSBs (52–54), and not RecF, RecJ, or RecQ (40–43). In agreement, we see RecB assurance and RecF adeptness of I–Sce I–induced RDG foci (Fig. 2E, DSB ). By contrast, ad-lib RecA/HR-dependent RDG/HJ foci anatomy apart of RecB (Fig. 3A), and were answer by RecF (Fig. 3A), advertence adjustment of non-DSB DNA damage. Further, accumulation of the phage Mu DSB end–binding protein Gam inhibits DSB adjustment (39) and I–Sce I–induced RDG foci (Fig. 3C) but did not affect ad-lib HR-dependent RDG foci (Fig. 3D and fig. S9, added controls). We achieve that the capital use of HJ-mediated HR in abundant (nonsexual) E. coli is adjustment of DNA accident added than DSBs. The RecF assurance implicates ssDNA gaps, a substrate at which RecF endless RecA (42, 43).

The ad-lib DNA accident that necessitates HR-repair HJs is replication-dependent. We blocked archetype admission at the E. coli origin, oriC, application a dnaATS allele, which allows archetype at 30°C but blocks oriC use at 42°C (55). We activate 30.8 ± 0.2 times beneath ad-lib RDG foci per aggregate of DNA (fig. S8G) in dnaATS than the WT ascendancy ache at the 42°C akin temperature (Fig. 3E, normalized to aggregate of DNA per cell; fig. S8G), but no aberration at acquiescent temperature (30°C). These abstracts abutment DNA archetype as the primary architect of the autogenous non-DSB DNA accident repaired by HJ-dependent HR commonly during abundant advance in E. coli. A archetypal for HR adjustment of replication-generated ssDNA gaps is discussed beneath (see Discussion).

We acclimated time-lapse microfluidic imaging to see the bearing and fates of ad-lib HJ foci in growing E. coli microcolonies. We empiric that best RDG/HJ foci persisted for at atomic 11 hours afterwards they appeared (93 ± 2%), and the beef in which they appeared accomplished to bisect (Fig. 3, F and G, 91 ± 5%). The HJs trapped by RDG ability anticipate chromosome segregation, which could block division, actuate a checkpoint, or both.

We activate that ad-lib HJ/RDG focus accretion was activated with corpuscle accommodation (Fig. 3, F and H), at ante from 0.033 ± 0.019 to 0.087 ± 0.018 foci per division, depending on corpuscle advance amount (Fig. 3I). The bearing assurance of best ad-lib HJs provides absolute abutment for DNA archetype as the disciplinarian of best ad-lib HR-HJ events.

Further, ad-lib HJs were activated with archetype forks. We bent chromosome numbers per corpuscle application breeze cytometry (fig. S8, D and E) (56), quantified ad-lib HJ/RDG foci via microscopy, and activate a about connected ad-lib RDG focus abundance per archetype fork: 5.0 × 10−3 (±0.3 × 10−3) and 4.2 × 10−3 (±0.6 × 10−3) in affluent and basal medium, appropriately (fig. S8F). Best beef with RDG foci had one focus per corpuscle (70.6 ± 0.4%), with the boyhood accepting added than one (fig. S8B). Because assorted HR adjustment contest appear as assorted foci per corpuscle (Fig. 2, A to D), we infer that best ad-lib adjustment HJs aftereffect from one or few DNA lesions per cell, rather than genome-wide catastrophe. The abstracts abutment DNA archetype as the abettor of best ad-lib DNA accident repaired by HJ-associated HR.

RecQ promotes both accretion and dissolution of HJs biochemically (16, 17, 57) and promotes net accretion of HR-HJs in beef (18); however, whether RecQ answer HJ formation, acceptable HR, or inhibited HJ resolution, abbreviation ad-lib HR in cells, was alien (18). We actualization that both RecQ DNA helicase and its accomplice RecJ ssDNA-dependent exonuclease advance ad-lib HR-HJ accretion and ad-lib HR in abundant E. coli cells.

We activate that ad-lib RDG foci were bargain by abatement of recJ or recQ, or recJ and recQ genes (Fig. 4A and figs. S9 and S10). We performed timed announcement studies to allurement HJs with RDG afore the accumulation of RecQ. HJ accretion proteins are accepted to access HJ levels apart of whether RDG is present aboriginal to allurement the HJs, admitting proteins that act afterwards HJ accretion (on HJs), such as RusA, are chock-full by the accumulation of RDG aboriginal (Fig. 1I). We activate that RecQ added RDG/HJ focus levels whether it was produced afore or afterwards RDG (Fig. 4B). These abstracts abutment RecQ and RecJ as promoters of HJ formation.

(A) RecQ and RecJ advance ad-lib HJ accretion acting in the aforementioned pathway. P = 0.002, P = 0.002, and P = 0.006, two-tailed unpaired t appraisal for recJ, recQ, and recJ recQ, respectively, compared with WT. (B) RecQ produced from an IPTG-inducible plasmid added RDG ad-lib HJ foci back produced afore (right) or afterwards (left) RDG. Larboard bar in anniversary panel, no IPTG induction. The abstracts announce that the RecQ advance of RDG foci after-effects from RecQ-promoted HJ formation, not RecQ-inhibiting HJ removal, which is blocked by RDG. (C) RecQ and RecJ advance ad-lib HJ/RDG foci via the RecA-dependent (HR repair) pathway, credible as no added abridgement of HJ/RDG foci in recA recQ or recA recJ bifold mutants above that in the recA distinct mutant. (D and E) RecQ and RecJ advance RecA-GFP focus formation, suggesting that they act afore RecA. (D) Representative images. Dejected arrows announce foci. (E) Quantification. Ad-lib RecA4155-GFP foci (WT) are bargain in recJ, recF, and recQ absent aberrant strains. P = 0.001, P = 0.004, and P = 0.02, two-tailed unpaired t appraisal (means ± SEM of three experiments, >1000 absolute beef denticulate in anniversary experiment). (F) RecA overproduction causes added credible non-HR–HJs, adjoin by RecQ and RecJ. RecA overproduction added RDG foci 2.09 ± 0.02 times (P = 0.01, two-tailed unpaired t test, pVector compared with pRecA; dejected bars), apart of RecF or RecB, implying a non-HR origin. There is no RecF assurance back the ad-lib HR-HJs are subtracted out (pRecA − pVector). Moreover, RecA overproduction added RDG foci by 11- and 9-fold in recQ- or recJ absent aberrant strains, appropriately (P = 0.001 and P = 0.0001, two-tailed unpaired t test). (G) The abundant access in RDG foci acquired by RecA overproduction in ΔrecQ beef is both RecF- and RecB-independent, implying that RecQ prevents non-HR–HJs. We advance that HJs are regressed archetype forks acquired by overproduced RecA (Fig. 6B).

Further acknowledging this conclusion, the afterward abstracts advance that RecQ and RecJ act afore RecA. We empiric that abatement of recQ or recJ from recA beef did not abate ad-lib HJs added than in recA beef (Fig. 4, A and C), advertence their action in the aforementioned alleyway as RecA. The ascertainment that ΔrecQ ΔrecA and ΔrecJ ΔrecA bifold mutants are added agnate to ΔrecA than to ΔrecQ or ΔrecJ distinct mutants (Fig. 4C) argues [per Avery and Wasserman (58)] that RecQ and RecJ act upstream of RecA in their pathway. We additionally saw that ad-lib foci of a aberrant RecA-GFP admixture protein (46) were bargain in ΔrecJ or ΔrecQ strains (Fig. 4, D and E). These abstracts betoken that RecQ and RecJ act afore RecA (strand exchange) in HR and may advance RecA loading assimilate ssDNA. RecF is the RecA loader at ssDNA gaps (42, 43), and 46 ± 11% of the ad-lib RecA-GFP foci were RecF-dependent (Fig. 4E), implying that these ad-lib RecA-GFP foci represent RecA on DNA at ssDNA gaps. We altercate a accessible pre-RecA role of RecQ and RecJ in postreplication ssDNA gap adjustment in the Discussion.

We advised ad-lib HR contest themselves to verify these conclusions. We activate that best ad-lib HR that recombined abutting chromosomal absolute echo sequences adapted RecF, RecQ, and RecJ (in accretion to RecA) (Fig. 5, A and B), adventurous a pro-HR role for RecQ, accordant with HJ accretion and adverse with inhibition of HJ resolution—the two agency that RecQ could accept answer net accretion of HR-HJs in beef (18). The RecF assurance of best ad-lib RDG foci (Fig. 3, A and B) and HR contest (Fig. 5B) supports recombinational ssDNA gap adjustment as the abettor of best ad-lib HJ foci in vegetative, growing E. coli beef (model, Discussion). The abstracts authenticate that RecQ and RecJ advance HR-HJ accretion in alive cells, and betoken that they act afore RecA during replication-induced ssDNA gap repair, and that this is a primary role commonly in growing cells.

(A) Strategy of the intrachromosomal absolute echo recombination appraisal of Corre et al. (82). (B) In beef afterwards bogus RecA overproduction, best ad-lib HR contest crave RecF, RecQ, and RecJ (and all of them crave RecA) and do not crave RecB (82). The abstracts announce that best of the ad-lib HR contest are instigated by ssDNA gaps [per Morimatsu and Kowalczykowski (42) and Sakai and Cox (43)], not, for example, RecFQJ-independent, RecB-dependent DSB adjustment (for example, Fig. 2E) (40, 41). These abstracts abutment the archetypal credible in Fig. 8. (C) Ad-lib HR contest are not added by RecA overproduction. This award suggests that the added RDG/HJ foci credible with overproduced RecA (Fig. 4, F and G) are not added HR-HJs, and supports the estimation that they reflect regressed archetype forks (illustrated in Fig. 6Biii). The baby abatement in ad-lib HR with overproduced RecA compared with the pVector ascendancy (P = 0.02, unpaired t test) could be acquired by titration of HJ resolution accommodation in the beef via their added non-HR–HJ levels (Fig. 4F). We accept that the RecQ and RecJ adeptness of the ad-lib HR contest in RecA-overproducing beef may announce that DSB-instigated HR may boss ad-lib contest during RecA overproduction. This is accepted because the attendance of added non-HR–HJs (probable RFs; Fig. 6Biii) is broken by RuvABC in alive beef bearing DSBs (33) and appropriately may annual DSB-instigated HR, which is RecQ- and RecJ-independent, to dominate.

The animal RecA ortholog RAD51 is overexpressed in a advanced ambit of tumors with p53 defects (11), and is activated with poor cast (9, 10); but how added RAD51 supports the blight accompaniment is unknown. We modeled RAD51 overexpression in cancers by overproducing RecA in E. coli and credible that RecA overproduction added HJ focus levels by a cogent 2.09 ± 0.02 times [Fig. 4F, pVector adjoin pRecA (blue bars)].

The afterward abstracts announce that the added RDG/HJ foci acquired by RecA overproduction are not HR-HJs but rather are non-HR–HJs, such as regressed archetype forks. Although RecA overproduction acquired added HJ foci (Fig. 4F), it did not access intrachromosomal HR contest in abutting absolute echo sequences (Fig. 5C), adventurous a abridgement of alternation amid HR and the added HJ foci observed.

In addition, the added RDG foci acquired by RecA overproduction were formed apart of RecA loader proteins that advance HR adjustment of DSBs, RecB (51), and ssDNA gaps, RecF (Fig. 4F) (42, 43). Note that ad-lib RDG foci are RecF-dependent (Fig. 4F, no abettor and pVector). If these are subtracted from the added RDG foci added by RecA overproduction (Fig. 4F, pRecA − pVector), again we see that all of the RecA overproduction–induced RDG foci are RecF-independent (Fig. 4F, pRecA − pVector). RecB and RecF are akin with animal BRCA2 (51–54, 59–61) and RAD52 (49). Because the RecF-dependent basic of ad-lib HR-HJ/RDG foci (Fig. 4F, pVector) remained present back RecA was overproduced (Fig. 4F, pRecA), and abandoned the added RDG/HJ foci are artless by RecF (Fig. 4F, pRecA − pVector), we can aphorism out the achievability that overproduction acquired RecA to become RecF-independent for HR. Therefore, RecA overproduction did not annual HR to become absolute of the HR RecA loader. Rather, the added HJs during RecA overproduction appear to appear by a different, non-HR process. We infer that the added HJs in this RAD51-overexpressing blight archetypal aftereffect from non-HR contest (uncorrelated with HR as illustrated in Fig. 5C, and absolute of RecA loaders as credible in Fig. 4, F and G): we advance from archetype angle blockage and regression, which occurs apart of RecF (35) and RecB (Fig. 6A) (33), and, biochemically, is answer by boundless RecA apart of loader proteins (35).

(A) Diagram of archetype angle blockage at a replication-blocking DNA bane (star) and corruption to anatomy a HJ. Back forks stall, the abeyant action of DNA supercoiling advanced of the angle drives the angle astern spontaneously (94), apart of RecA biochemically (94) and in beef (33). Lines, strands of DNA; alongside lines, base-paired strands; arrowheads, 3′ ends. (B) Archetypal for angle corruption acquired by balance RecA and its blockage by RecQ/RecJ. We advance that with RecA overproduction, forks backslide spuriously, afterwards a replication-blocking DNA lesion, because of RecA polymerization on ssDNA at the angle (i and ii) instigating fiber barter with the beginning sister duplex. (ii) RecA can be loaded on ssDNA at a angle and connected 3′ from the angle alliance to the ssDNA-dsDNA (double-stranded DNA) alliance at the 3′ arch fiber end, a RecF-independent (35) [and RecB-independent (33)] absolute substrate and acknowledgment to advance (iii) angle regression. We advance that afterwards RecA loading assimilate the ssDNA, there can be two outcomes, which are afflicted by the attendance of RecQ or RecJ. (iii) In the absence of RecQ or RecJ, RF HJs will accumulate. (iv) In the attendance of RecQ and RecJ, RecQ unwinding of the backward fiber at the angle (19, 69) and RecJ 5′-ssDNA–dependent exonuclease can anticipate some RF HJs by authoritative a ssDNA-end angle corruption structure/substrate. This 3′-ssDNA end (arrowhead) would not be apprenticed by RDG and may be abject by ssDNA exonucleases, preventing HJs.

Surprisingly, we activate that RecA overproduction resulted in RecQ and RecJ roles adverse to their roles in ad-lib HR (Fig. 5B) and HR-RDG focus accretion (Fig. 4, A to E); RecQ and RecJ adjoin accretion of the added non-HR–HJs acquired by overproduced RecA (Fig. 4F). Beef that abridgement RecQ or RecJ (ΔrecQ or ΔrecJ) showed 11.1 ± 0.2 and 8.8 ± 0.1 times added RDG foci/HJs acquired by RecA overproduction, appropriately (Fig. 4F). The boundless RDG foci/HJs in ΔrecQ beef are additionally absolute of the RecA loaders RecB and RecF (42, 43), which are acclimated in HR adjustment (Fig. 4G). Moreover, we induced RDG, which accessories HJs, afore RecA overproduction (fig. S11). Therefore, the abstracts announce that RecJ and RecQ prevented the formation, rather than aided the postformation removal, of the non-HR–HJs acquired by RecA overproduction. The RecQ/J role(s) in preventing non-HR–HJs (Fig. 4F) contrasts with the RecQ and RecJ roles in announcement HR-HJ accretion that dominates ad-lib HJ contest (Figs. 4, A to E, and 5B; see archetypal in Discussion).

Thus, RecQ plays a atypical junction-guardian role in E. coli: both announcement ad-lib HR (repair)–HJs and preventing credible non-HR–HJs. RecQ/J may anticipate the accretion of non-HR–HJs acquired by archetype blockage and angle corruption (model, Fig. 6B). We advance that overproduced RecA promotes regressed-fork (FR), RF HJs abnormally by acting on absolute (normal) forks (model, Fig. 6B), rather than aloof those with replication-blocking accident (star, Fig. 6A). We achieve that RecQ/J comedy a atypical junction-guardian role in preventing non-HR–HJs that aftereffect from RecA overproduction (model, Fig. 6B) while accompanying announcement HR adjustment HJs in ssDNA gap adjustment (model, Discussion).

We looked for RNA abstracts correlations that ability announce animal RecQ orthologs with abeyant agnate junction-guardian roles in RAD51-overexpressing cancers. We accepted that if the RecQ junction-guardian action improves the fitness/growth of RecA-overproducing cells, again RAD51-overexpressing cancers ability crave proteins that comedy RecQ-like roles in preventing or abbreviation balance HJs acquired by RAD51 overproduction. Thus, the genes encoding proteins with RecQ-like or HJ resolvase functions ability be overexpressed in RAD51-overexpressing cancers. We activate that the animal RecQ orthologs BLM and RECQL4, as able-bodied as a HJ endonuclease gene, EME1, are frequently co-overexpressed with RAD51 in the eight best accepted cancers for BLM and in four of the eight best accepted cancers for RECQL4 (Spearman’s alternation appraisal credible in Fig 7, A to E, and table S1).

Spearman’s rank alternation analyses of abstracts from cBioPortal (88, 89), per Abstracts and Methods. Anniversary abstracts point represents the mRNA akin in one accommodating sample about to the advertence citizenry (Z score; Abstracts and Methods). Abstracts from 129 to 1100 accommodating samples were analyzed per blight blazon (table S1). (A) Added BLM mRNA levels (y axis, Z scores) activated with added RAD51 mRNA (x axis, Z scores) in eight of eight of the best accepted blight types (R2 > 0.25; P ≤ 6.23 × 10−21, Spearman’s rank alternation analysis). (B) Added RECLQ4 mRNA levels (y axis, Z scores) activated with added RAD51 mRNA (x axis, Z scores) in four of eight of the best accepted blight types (R2 > 0.25; P ≤ 2.07 × 10−8, Spearman’s rank alternation analysis). (C) Summary: alternation of added RAD51 mRNA levels with added levels of BLM and RECLQ4 RecQ orthologs, EME1 HJ resolution protein, and added RecQ orthologs and resolution proteins in accommodating bump abstracts of the eight best accepted cancers (numbers in parentheses, cardinal of accepted cancers of the eight best accepted types with correlation). mRNA Spearman’s alternation accessory affected amid these and added animal RecQ orthologs and added HJ resolvases with RAD51 and with anniversary added amid the eight best accepted cancers is abbreviated in table S1 (R2 > 0.25 indicates abstinent correlation; see table S1 for details). (D) Summary of Spearman’s R2 ethics for blight RNAs of genes activated with RAD51 overexpression (R2 > 0.25 for abstinent correlation) and ascendancy genes ailing activated or uncorrelated with RAD51 overexpression (for example, ACTB, which encodes a subunit of actin).

In both R2 ethics and numbers of blight types involved, these actual able correlations (table S1) outscored accepted transcriptional alternation studies advised to be awful apocalyptic to definitive. For example, CDH1 and ZEB1 (62), and KLF4 and KLF5 (63), showed R2 ethics of 0.15 and 0.08, both in abandoned a distinct blight type, compared with the R2 ethics for BLM with RAD51 (0.29 to 0.62) in the eight best accepted blight types, and for RECQL4 with RAD51 (0.26 to 0.41) in four of the eight best accepted blight types (Fig. 7, A to E). Thus, these are acutely able-bodied correlations. Negative ascendancy and added genes did not actualization such correlations (for example, ACTB encoding a subunit of actin; Fig. 7D).

We accept that the added announcement of the RecQ orthologs BLM and RECQL4 and the HJ resolvase EME1 may additionally anticipate (or remove) balance HJs. The best coexpressed HJ resolvases and RecQ ortholog with BLM are EME1, GEN1, and RECQL4 (Spearman’s alternation appraisal credible in fig. S12 and table S1). These correlations may announce that EME1, GEN1, and RECQL4 may assignment calm with BLM to abate or anticipate balance HJs acquired by RAD51 overexpression, a accepted accident in abounding and assorted cancers. Added assignment is bare to appraisal these hypotheses.

We showed that RuvC-derived, catalytically abeyant beaming protein fusions (RDG, green; RDM, red) accurately bind, trap, and characterization four-way DNA junctions (HJs), acceptance their altitude as beaming foci in distinct alive beef (Figs. 1I, 2, A to F, and 3 and figs. S7 and S8) and their mapping in genomes via ChIP-seq (Fig. 2, G and H, and fig. S6). RDG binds and protects HJs from the action of added proteins, that is, accessories HJs, back antiseptic in band-aid (Fig. 1E and fig. S2) and in alive beef (Figs. 1, F to I, and 2). The estimated adeptness of apprehension of HJs (or bifold HJs) as alone beaming foci is almost 50% (text S2). Like the engineered DSB-trap proteins created advanced (39), RDG and RDM are advantageous accoutrement for belief DNA acknowledgment intermediates in distinct alive beef and in genomes.

A limitation of RuvC-based HJ accessories is their assurance on E. coli RuvB (Figs. 2E and 3, A and B), apparently via their specific protein-protein interactions (32). We are currently exploring whether this will absolute the capability of these accoutrement in added organisms.

Although DSB adjustment mechanisms are the best advised HR reactions, we acquisition that the capital use of HJ-associated HR in abundant (nonsexual) E. coli beef is not DSB repair. Surprisingly, ad-lib HJs are (i) 75% HR-based (Fig. 3, A and B), (ii) DSB-independent (Fig. 3, C and D), (iii) mostly RecFJQ-dependent (Figs. 3, A and B, and 4, A to C), (iv) replication-dependent (Fig. 3E), (v) activated with archetype angle numbers (Fig. 3, F to H, and fig. S8), and (vi) about 100 times beneath accepted than the already per E. coli genome archetype estimated advanced (Fig. 3, F to H; fig. S8; and argument S2) (14, 40). Collectively, our abstracts betoken that adjustment of replication-induced ssDNA gaps (illustrated in Fig. 8) is the commonest use of HR and antecedent of HJ intermediates in growing E. coli cells. Autogenous DNA accident is the primary agitator of repair-based genomic changes that drive cancer, abiogenetic diseases, and microbial change (see Introduction). We advance that single-strand lesions aggregate the primary agitator of those changes. Added ecology altitude ability aftermath added results.

This archetypal follows the annual of Rupp and Howard-Flanders (95) and integrates the abstracts (i) that best ad-lib RDG/HJ foci and intrachromosomal HR contest are RecA- and RecF-dependent HR-HJs (Figs. 3, A and B, and 5B and fig. S10), absorption RecA loading at single-strand gaps, and abundantly RecQ- and RecJ-dependent (Figs. 4, A to C, and 5B and fig. S10), with RecQ/J acting in the RecA-dependent alleyway (Fig. 4C); (ii) that RecQ/J act afore HJ formation, credible by their advance of HJs alike afterwards RDG is produced (Fig. 4B); and (iii) that RecQ/J advance credible RecA loading in assays of ad-lib RecA-GFP foci (Fig. 4, D and E) anticipation to reflect RecA-DNA nucleoprotein filaments (46). Model: During abundant advance and DNA replication, (A) back a amalgam amplitude of backward fiber DNA (an Okazaki fragment, arrowheads, 3′ ends) encounters a replication-blocking bane (star) in its arrangement strand, (B) the adjoining Okazaki fragment can be unwound by RecQ DNA helicase, which translocates on DNA in the 3′ to 5′ administration as credible by Hishida et al. (69), advertisement a 5′-ssDNA end, which is again abject by RecJ 5′-ssDNA–dependent exonuclease. This creates a single-stranded gap, which can be coated by SSB [E. coli single-stranded DNA bounden protein, like animal archetype protein A (96)]. (C) RecF is guided to a 5′ end of a ssDNA-dsDNA alliance (43) and endless RecA assimilate SSB-coated ssDNA (42). The RecA-DNA nucleoprotein circuitous again promotes fiber barter (40, 41) so that the blocked 3′ Okazaki fragment end can displace the identical arrangement (black line) in the sister chromosome, and prime connected DNA amalgam (dashed red line) application the new fiber of the sister as a template. Lines, strands of DNA; red lines, new DNA strands; atramentous lines, old strands; abject red line, new DNA amalgam afterwards fiber exchange. (D) Annex clearing of the HJ rightward, accomplished the lesion, places anew actinic DNA beyond from the lesion. Added annex rightward allotment the anew actinic fiber to its aboriginal arrangement (E) and allotment the 3′ end to the aboriginal bifold (F), removing the HJ and D loop. The bane is bypassed and, already it is allotment of dsDNA, can be repaired by abatement adjustment pathways, which can act abandoned back the damaged abject or nucleotide is present in dsDNA [base abatement adjustment and nucleotide abatement repair; advised by Friedberg et al. (97)]. An accretion approach of resolution of the HJ is endonucleolytic cleavage, for example, by RuvC (not shown).

When DSBs do occur, as back induced by I–Sce I endonuclease, their adjustment shows affirmation of two modes of whole-genome ascendancy and directionality forth the chromosome (Fig. 2, G and H, and fig. S6). First, I–Sce I–induced, RecA- and RecB-dependent HR-HJs occurred not abandoned at the acclimation DSB sites (between 60 and 200 kb on either side) but additionally at the chromosomal archetype abuttals region, megabases abroad (Fig. 2, G and H, and fig. S6). These abstracts abutment a replicative archetypal for HR-DSB adjustment (64), in which archetype forks are abreast by HR strand-exchange intermediates (replication restart at a DSB) that accommodate a HJ (Fig. 9C). The abstracts advance that the archetype accomplished by HR-DSB adjustment [break-induced archetype (BIR)] continues from the DSB to the archetype abuttals (Fig. 9) (65), and betoken that HR-initiated archetype bubbles can sometimes annoyance their associated HJs from the DSB (65) all the way to the archetype abuttals (Fig. 9E), which is a new discovery. DSB adjustment in E. coli is abundantly abased on the above replicative DNA polymerase, Pol III (65), and produces conservatively absolute new strands of DNA (65), acknowledging the archetypal in Fig. 9, adapted from the archetypal adapted by Motamedi et al. (65). In yeast, BIR balloon clearing was empiric and anticipation to reflect D loops afterwards HJs (similar to Fig. 1Aiii) (66), but we advance that it ability also, at atomic sometimes, accommodate changing HJs, as we accept empiric actuality (Fig. 2, G and H, and fig. S6). These abstracts abutment both the replicative attributes of abundant of DSB adjustment and the accepted aberration (one-endedness) of the events, which allows connected genomic archetype (for example, in adverse with two-ended mechanisms such as credible in Fig. 1A). Alternatively, the DSB repair–instigated HJs at the archetype abuttals ability aftereffect from site-specific HJ-dependent resolution of chromosome dimers acquired by crossing-over during HR-DSB repair, which is performed by the XerCD site-specific recombinase at the archetype abuttals dif armpit [reviewed by Lesterlin et al. (67)]. This seems beneath acceptable because the accessory of terminus-proximal reads in ChIP-seq was far broader and beyond than the 29–base brace (bp) dif site, to which XerCD site-specific HJs are confined.

(A) to (C) absorb annual from Kuzminov (68), and (D) and (E) allegorize a archetypal of Motamedi et al. (65). (A) Chi sites abate RecBCD double-strand exonuclease action (40, 41) and are anchored abnormally in the genome such that if a two-ended DSB were to occur, (B) the DNA on the archetype terminus–proximal ancillary would be acceptable to be abject abundantly (few alive Chi’s in the ori-to-ter direction), admitting the DNA on the ori-proximal ancillary would ache beneath abasement (many alive Chi’s in the ter-to-ori direction) (68). (C) Thus, the nondegraded DSB end would admit HR adjustment by fiber barter that would prime a archetype angle that would run in the chromosome’s accustomed ori-to-ter administration adjoin the terminus. (D and E) The archetypal genitalia illustrated in (C) to (E) were offered advanced by Motamedi et al. (65) in abutment of our observations that best DSB adjustment via HR requires the above replicative DNA polymerase (Pol III) and that the new strands are absolute conservatively (65), as empiric afterwards in aggrandize BIR (98, 99). We adapted that the archetype balloon gain adjoin the terminus, boring an changing HJ abaft it, which armament the new DNA strands out of the bubble, causing the empiric bourgeois allegory of anew actinic DNA strands in DSB adjustment archetype (65). (E) With their abaft HJs, forks that activate on one ancillary of the abuttals will abeyance and accrue at the terminus. Some that overshoot the amid point will be chock-full at the unidirectional ter sites(s) (red, triangular abandon stop advancing forks) on the adverse ancillary of the chromosome centermost point from which the balloon began. This arrangement of HJ accretion is credible in the ChIP-seq abstracts credible in Fig. 2 (G and H).

Second, the abstracts actualization skewed distributions of HJs about acclimation two-ended DSBs in the E. coli chromosome with added HJs on the terminus-proximal ancillary of the DSB than on the ori-proximal ancillary of the DSB. That is, there are added HJs afterwards than upstream of the DSB in the chromosomal archetype paths (Fig. 2, G and H, and fig. S6) or “replichores.” For two altered I–Sce I breach sites, there were added RecA- and RecB-dependent HJs terminus-proximally than ori-proximally (Fig. 2, G and H, and fig. S6). These abstracts abutment the Kuzminov archetypal (68) of absurd DNA abasement and adjustment at DSBs in the E. coli chromosome (illustrated in Fig. 9, A to C) controlled by the absurd administration of chromosomal Chi sites. Chi sites abate RecBCD dsDNA exonuclease action to acquiesce HR end adjustment (40, 41) and abatement abnormally in the E. coli genome with added alive Chi sites upstream than afterwards from any point in the replichores. Therefore, at any DSB in the genome, the ori-proximal DNA end is bigger adequate from abasement by RecBCD exonuclease (more alive Chi’s) and is added acceptable to be preserved, admitting the terminus-proximal DSB end is added acceptable to be abject (few alive Chi’s), credible in Fig. 9B (68). Preservation of the ori-proximal end and accident of the ter-proximal end (Fig. 9B) were proposed to annual one-ended DSB adjustment archetype (BIR), with the administration of the adjustment archetype preferentially from ori (the preserved end that initiates HR-mediated archetype restart; Fig. 9, B and C) adjoin the abuttals (Fig. 9, B to E). This archetypal is astute but untested. Our ascertainment of added DSB-induced adjustment HJs terminus-proximally to DSBs (Fig. 2, G and H, and fig. S6) is predicted by the Kuzminov one-ended adjustment archetypal if the HJs that accompany the fiber barter that primes BIR sometimes abide with the archetype bubble, unresolved, and biking with the balloon adjoin the abuttals (Fig. 9, C to E). The bent adjoin HJs ori-distally from DSBs ability alternatively reflect two-ended DSB adjustment HJs (not credible in Fig. 9, but agnate to that in Fig. 1A, iii and iv) afterward absurd abasement (Fig. 9B). Added Chi-independent absurd chromosomal actualization ability underlie the HJ aberration empiric (replication forks in progress, ascendant admonition of transcription). Added assignment is bare to analyze specific models. Behindhand of the specific mechanism, the abstracts authenticate genome-scale ascendancy and directionality of DSB adjustment contest forth the E. coli genome.

We credible a atypical junction-guardian role for E. coli RecQ DNA helicase, ortholog of bristles animal blight blockage proteins (15), in announcement and preventing HJs. First, RecQ appears to act upstream of RecA loading in alive beef (Fig. 4, D and E) and is adapted for the accretion of best RecA-dependent ad-lib chromosomal HR-HJs (Fig. 4, A to C) and additionally for best ad-lib intrachromosomal HR contest (Fig. 5B). Our abstracts announce that RecQ promotes HR-HJs during replication-instigated ssDNA gap adjustment (model illustrated in Fig. 8). Second, we additionally credible that RecQ and RecJ anticipate non-HR–HJs induced by RecA overproduction (Figs. 4, F and G, and 5C; archetypal illustrated in Fig. 6B), an E. coli archetypal of abounding p53-defective cancers with RAD51 overexpression (11). Our after-effects are partly agnate to antecedent ascertainment of UV light–induced accretion of plasmid DNA structures articular by aggregate two-dimensional gel electrophoresis as either double-Y junctions or HJs (20), which, like our non-HR–HJ foci, were adjoin by RecQ and RecJ but, clashing our after-effects (Fig. 4, F and G), were abased on RecF (20). Admitting those authors adapted a post-HJ role of RecQ and RecJ in removing regressed archetype angle HJs, our abstracts betoken that RecQ and RecJ anticipate the accretion of (RecF-independent) RF HJs. That these non-HR–HJs are induced by overproduction of RecA models abounding p53-defective cancers with RAD51 overexpression (11).

RAD51 overexpression supports breast blight metastases (9) and is activated with decreased adaptation of lung blight patients (10). We credible that RecA overproduction decidedly added HR protein–independent HJs and that RecQ/J comedy a atypical junction-guardian role in preventing these (Fig. 4, F and G; model, Fig. 6B). RecA overproduction did not annual RecF adeptness of HR (Fig. 4F), and HR was not activated with added RecA-induced HJs (Fig. 5C), such that best of the RecA overproduction–induced added HJs (Fig. 4, F and G) are alive to appear HR-independently. We advance that these non-HR–HJs are regressed archetype forks (Fig. 6Biii). We accept that balance RecA/RAD51 causes angle corruption spuriously on absolute DNA (illustrated in Fig. 6B). RecQ and RecJ could anticipate the HJ date of angle corruption by unwinding and digesting the backward strand, as credible (Fig. 6Biv) and alive by RecQ and RecJ biochemistry (69) and DNA abasement in beef (19, 70, 71). This dual, junction-guardian role (promoting HR-HJs, preventing RF-HJs) could be aggregate by one or added animal RecQ orthologs. Acknowledging this hypothesis, we activate that BLM and RECQL4 are co-overexpressed with RAD51 in the eight and in four of the eight best accepted blight types, appropriately (Fig. 7, fig. S12, and table S1). Moreover, the accepted animal HJ resolution protein genes EME1 and GEN1 (72) are co–up-regulated with BLM and RAD51 (Fig. 7, fig. S12, and table S1). We advance that at atomic one aftereffect of overexpressed RAD51 in tumors could be added RFs, which are genome-destabilizing and annual genome change that could drive the blight accompaniment (73), and that BLM and RECQL4 may anticipate RF-HJs, admitting EME1 and GEN1 may carve the RFs, creating DSB ends that aeon through adjustment and replication. Antiseptic BLM can advance angle corruption in band-aid (74), and RECQL4 promotes HR-HJs (26). Perhaps they additionally anticipate RF-HJs in cells. Failure to abolish RFs would block archetype and chromosome segregation, so their abatement would be accepted to be called in cancers. Added assignment is bare to appraisal these hypotheses. We are alive adjoin animal cell–compatible HJ accessories that may aid analysis of these and abounding added annual apropos HJs and genome alternation in animal cancers.

Strains acclimated in this abstraction are accustomed in table S2. Bacteria were developed in Luria-Bertani-Herskowitz (LBH) affluent average or M9 basal average (75) supplemented with thiamine (10 μg/ml; vitamin B1) and 0.1% glucose or glycerol as a carbon source. Added additives were acclimated at the afterward concentrations: ampicillin (100 μg/ml), chloramphenicol (25 μg/ml), kanamycin (50 μg/ml), tetracycline (10 μg/ml), and sodium citrate (20 mM). P1 transductions were performed as declared by Thomason et al. (76). Genotypes were absolute by antibacterial resistance, polymerase alternation acknowledgment (PCR), and, back relevant, UV acuteness and sequencing.

The ruvC gene (without stop codon) and gfpmut3 (77) (or mCherry) (39) gene were alloyed with a bond arrangement (5′-GCTATCGACGAAAACAAACAGAAAGCGTTGGCGGCAGCA-3′) application the agreement declared by Heckman and Pease (78) and ligated into the pET28a vector. The two mutations (D7N and E66D) of ruvCDef were generated by site-directed mutagenesis (Stratagene). The doxycycline-inducible PN25tetO apostle from pRF3 (39) was subcloned upstream of ruvC (or ruvCDefgfp or ruvCDefmCherry) to alter the T7 apostle of pET28a. A chloramphenicol attrition (cat) cassette belted by FRT sites from pKD3 (79) was subcloned afterwards from ruvC (or ruvCDefgfp or ruvCDefmCherry). To move the absolute announcement arrangement into the chromosome application Red-mediated short-homology recombination (79), we amid genitalia of mntH and nupC genes (~1 kb) upstream and afterwards of PN25tetOruvC (or ruvCDefgfp or ruvCDefmCherry) as akin sequences, respectively. A apostle PN25tetO-only accumulate was additionally confused into the chromosome as a control. The completed plasmid constructs are listed in table S3 (pLC1 to pLC4). Anniversary absolute announcement cassette was confused abandoned into the chromosome of SMR19152 by Red-mediated recombineering application primers P1 and P2 (table S4) and again transduced into the strains listed in table S2. SMR19152 encodes constitutively produced TetR protein that represses the PN25tetO apostle in the absence of the inducer doxycycline (80). All constructs were absolute by PCR, antibacterial resistance, and sequencing.

The ruvCgfp and ruvCDefgfp genes were subcloned into pET28a announcement vectors and again adapted into the BL21 acquired ache (T7 Express lysY/Iq Competent E. coli, New England Biolabs). Afterwards consecration with 0.1 mM IPTG, the proteins with an N-terminal His6-tag were produced at 20°C for 16 hours and pelleted by centrifugation at 7000 rpm for 30 min. The corpuscle pellet was resuspended in lysis absorber [50 mM Hepes (pH 8.0), 300 mM NaCl, 5 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride] and disrupted by sonication (10 times for 30 s, on/off at achievement 5; XL-2000, Misonix). The absolute corpuscle lysate was centrifuged at 30,000g for 40 min, and the acrid recombinant protein was antiseptic by anchored metal ion chromatography with a Ni-NTA cavalcade (Qiagen). Afterwards abrasion with ablution absorber (lysis absorber with 50 mM imidazole added), the protein was eluted with elution absorber (lysis absorber with 500 mM imidazole) and added desalted adjoin the accumulator absorber [20 mM Hepes (pH 8.0) and 20 mM NaCl]. The protein was concentrated to about 2 mg/ml and stored at −80°C. The Flp protein was antiseptic to abreast accord application DNA affection accessory as the final footfall (81).

Proteins were afar by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) film according to the manufacturer’s instructions (Amersham, GE Healthcare). The membranes were blocked with ECL Prime blocking abettor (GE Healthcare) and probed with primary abrasion anti-RuvC monoclonal immunoglobulin G (IgG) antibodies (Santa Cruz Biotechnology). The film was added probed with accessory polyclonal dupe anti-IgG antibacterial (Bethyl Laboratories) and visualized by scanning in a checkered imager Typhoon apprehension arrangement (GE Healthcare).

Saturated overnight, LBH cultures were adulterated 100-fold in LBH average and developed at 37°C for 1.5 hours, at which time doxycycline (100 ng/ml) was added to abet protein production. Afterwards 1 hour of induction, beef were argent on LBH solid average absolute doxycycline (10 ng/ml), and the argent beef were ablaze with altered doses of UVC ablaze application a Stratalinker 1800 UV crosslinker (Stratagene) and again incubated in the aphotic brief at 37°C for antecedents quantification. Ascendancy cultures afterwards doxycycline consecration were advised identically.

The oligonucleotides acclimated for the accumulation of HJs (HJa-HJc) that served as substrates in bounden and breach assays (Fig. 1E and fig. S2, A to C) are listed in table S4. For HJa, accumulated from oligonucleotides HJa1 to HJa4, the bases that anatomy a 2-bp affinity amount in the axial arena of HJ for RuvC breach are credible in bold. For HJb, composed of oligonucleotides HJb1 to HJb4, the bases that anatomy the acceptance arrangement for Flp are credible in bold. HJc, complete from oligonucleotides HJc1 to HJc4, was the substrate for brake agitator assimilation assays. The sequences that anatomy the acceptance sites for brake enzymes in HJc are credible in bold. The four accoutrements of HJb were according in length, as were those of HJc, and amidst an anchored annex point. To accumulate a accustomed junction, equimolar amounts of the four requisite oligonucleotides were accumulated in 50 mM tris-HCl (pH 8.0), 5 mM EDTA, and 1 mM dithiothreitol (DTT), and the admixture was placed for 5 min in a baptize ablution maintained at 80°C. Heat was angry off, and the ablution was accustomed to air-conditioned boring to allowance temperature on the bank top. About quantitative accumulation of the alliance was absolute by gel electrophoresis.

The bounden and antagonism assays were performed in 50 mM tris-HCl (pH 8.0), 5 mM EDTA, 1 mM DTT, 5% glycerol, and bovine serum albumin (100 mg/ml), absolute 2 pmol of the HJb per evolution mixture. RuvCGFP or RDG gave complete bounden of the alliance at a molar arrangement of 1:10 (junction to protein). For the accepted bounden assays, incubations were agitated out on ice for 15 min afterwards protein addition. For the antagonism assays, the alliance was preincubated with RDG on ice for 15 min (1:10, alliance to protein) afore accretion of Flp (1:4 or 1:8, alliance to protein) and incubated for an added 15 min. The bounden mixtures were fractionated by electrophoresis in a 3% agarose gel in 6.7 mM tris-HCl (pH 8.0), 3.3 mM sodium acetate, and 2 mM EDTA at 4°C for 75 min (10 V cm−1) with connected recirculation of the buffer. The DNA bands were visualized beneath UV ablaze afterwards staining the gel with ethidium bromide. In some assays, the junction-bound proteins were electrotransferred to a PVDF film application a semidry alteration corpuscle (Bio-Rad, 170-3940) and probed with antibodies to GFP (to ascertain RDG) or with antibodies to built-in Flp. The apprehension acknowledgment acclimated peroxidase-conjugated accessory antibodies (anti-mouse for GFP; anti-rabbit for Flp) in affiliation with an added chemiluminescence-based substrate (Pierce). A abrasion monoclonal antibacterial to GFP was purchased from Abcam. A polyclonal antibacterial to Flp was aloft in rabbits adjoin a complete Flp peptide. For protein detection, the GFP and Flp antibodies were adulterated 1:1000 and 1:5000, respectively.

The acknowledgment mixtures (20 μl) absolute 0.25 μM HJa and 1 μM antiseptic protein in 20 mM Hepes (pH 8), 10 mM MgCl2, and 5% glycerol were incubated for 1 hour at 37°C afore the accretion of 5 μl of endlessly absorber [100 mM EDTA (pH 8), 1% SDS, 20% glycerol, 0.1% bromophenol blue, and proteinase K (5 μg/μl)] followed by evolution at 55°C for 30 min. The samples were analyzed by 3% agarose gel electrophoresis at 100 V.

The DNA substrate was a HJ absolute the Eco RI acceptance arrangement in one of the arms. Assimilation was performed on the chargeless alliance or afterwards bounden by RuvCGFP or RDG. Anniversary acknowledgment admixture absolute 2 pmol of the alliance and 2.5 U of Eco RI (New England Biolabs). Prebinding of the alliance by RuvCGFP or RDG was agitated out in the assimilation absorber (supplied by New England Biolabs) for 10 min on ice afore agitator addition. The molar arrangement of the alliance to RuvCGFP or RDG was 1:10. Immediately afterward agitator addition, the acknowledgment mixtures were transferred to a 37°C baptize bath. Reactions were chock-full by the accretion of 0.2% SDS, and samples were analyzed by electrophoresis in 3% agarose gel at allowance temperature (10 V cm−1). DNA was visualized by ethidium boiler staining. Altitude in plots shows agency ± SEM from three absolute experiments.

DNA bands from gels were analyzed application Quantity One software from Bio-Rad.

Quantitative P1 transduction assays were as declared by Magner et al. (18), with the modification that saturated brief aqueous LBH cultures were adulterated 100 times in LBH average with or afterwards doxycycline (100 ng/ml) to abet RDG and developed at 37°C for 3 hours. Phage P1 developed on ache SMR6263 was acclimated to transduce leu::Tn10 into almsman strains. The transductants were argent on the Tet plates with or afterwards doxycycline to abet RDG. The frequencies of TetR transductants were bent by three P1 transductions with almsman beef in balance (multiplicities of infection of <0.01 phage per almsman cell). The absolute echo HR appraisal was as declared by Corre et al. (82) with the modification that some of the isogenic strains additionally backpack plasmid abettor or isogenic RecA-overproducing plasmid and were or were not induced to overproduce RecA by abacus 1 mM IPTG.

Images were visualized with an astern DeltaVision Amount Image Restoration Microscope (GE Healthcare) with a 100× UPlan S Apochromat (numerical aperture, 1.4) cold lens (Olympus) and a CoolSNAP HQ2 camera (Photometrics). Captured images were called about beneath the microscope. The images were taken with Z endless (0.15-μm intervals) and again deconvoluted (DeltaVision SoftWoRx software) to see the accomplished corpuscle for absolute altitude of foci in anniversary cell. For anniversary absolute experiment, >1000 beef (about 3000 beef for ΔrecA and ΔrecB strains) were counted application ImageJ software with beheld analysis in anniversary of three absolute experiments.

For the altitude of ad-lib HJ focus formation, saturated brief cultures were adulterated 1:100 in beginning LBH average or 1:25 concoction in M9-glucose average and incubated at 37°C for 1.5 hours afore abacus doxycycline (100 ng/ml). Cultures were incubated for accretion 4 hours afore visualizing beneath the microscope. Advance curves of altered ache backgrounds were agnate (fig. S9). For I–Sce I–induced foci, saturated brief cultures started in average absolute 0.1% glucose were adulterated 1:100 or 1:25 in beginning LBH or M9-glycerol average supplied with proline (50 μg/ml), respectively, and incubated at 37°C for 1.5 hours afore abacus doxycycline (100 ng/ml). Afterwards 1 hour, 0.005% arabinose was added and evolution was connected for accretion 3 hours. The action of γ-radiation–induced focus accretion was performed as advanced declared by Shee et al. (39), except for application altered doses of γ-radiation.

For abstracts with temperature-inducible Gam production, cultures were inoculated into affluent average from saturated brief cultures at 30°C for 1.5 hours afore alive to 37°C (to abet Gam production) and abacus doxycycline (100 ng/ml; to abet RDG). Ad-lib foci were counted afterwards accretion 4 hours of incubation. For Gam accumulation beneath DSB-inducing conditions, the temperature was confused and doxycycline was added for 1 hour afore abacus arabinose to abet I–Sce I to accomplish DSBs. Foci were counted afterwards accretion 3 hours of evolution at 37°C. RecA was overproduced by abacus 1 mM IPTG. For dnaATS temperature about-face experiments, cultures were inoculated into affluent average from saturated brief cultures at 30°C for 1.5 hours afore alive to 42°C (to inactivate DnaA) and abacus doxycycline (100 ng/ml; to abet RDG). Ad-lib foci were counted afterwards accretion 4 hours of incubation.

The action was as declared by Shee et al. (39), except for a slight aberration in advance condition. Saturated cultures of SMR19382 were adulterated 100-fold in M9 glucose average supplemented with vitamin B1, 0.5% casamino acid, and doxycycline (10 ng/ml) and developed at 37°C for 1 hour afore loading beef into the microfluidic alcove (time 0). For the abutting 7 hours, beef were bathed with the aforementioned medium, but with abandoned doxycycline (2 ng/ml) to acquiesce division, again switched at 7 hours to the aforementioned average afterwards glucose, and bathed until 18 hours. The cardinal of corpuscle accommodation and the actualization of RDG foci were captured application time-lapse diminutive photography throughout the experiment.

Cell cultures were developed in LBH with 0.1% glucose to assimilation overnight, again adulterated 100-fold into 100 ml of LBH in 500-ml flasks, and incubated at 37°C for 2.5 hours afore the accretion of doxycycline (100 ng/ml) to abet RDG production. Afterwards 30 min, 0.005% arabinose was added to abet I–Sce I expression. Afterwards accretion 2.5 hours of evolution at 37°C, proteins and DNA were cross-linked by the accretion of 1% formaldehyde for 30 min at allowance temperature. The cross-linking was quenched by the accretion of 0.5 M glycine, and beef were harvested by centrifugation and done already with tris-buffered saline. Beef were lysed as declared by Bonocora and Wade (83) with lysis absorber absolute lysozyme (4 mg/ml). Lysates were sonicated application the Bioruptor Pico (Diagenode) until best of the DNA bits were amid 300 and 500 bp. Ribonuclease A was added to annihilate RNA-related interactions. The immunoprecipitation and library alertness were performed at the aforementioned time as declared by Bonocora and Wade (83). Sequencing was performed on an Illumina MiSeq.

Cultures were developed as declared in ChIP-seq library preparation, except that afore that protocol’s cross-linking step, beef were calm by centrifugation and genomic DNA was extracted and antiseptic application the DNeasy Blood & Tissue Kit (Qiagen). Libraries were able application Nextera XT kits (Illumina), and sequencing was performed on an Illumina MiSeq.

Before mapping, FASTX-Toolkit (v0.0.14) was acclimated to preprocess the sequences: Adaptor sequences were removed, and reads were akin and filtered according to quality. The arrangement alignment was performed by BWA-MEM (v0.7.12) with –M advantage to mark beneath breach hits as accessory alignments and added ambit as absence (84). Depending on the ache background, reads were mapped to either the W3110 genome [National Centermost for Biotechnology Information (NCBI) Advertence Arrangement (RefSeq) Database accession: NC_007779.1] or the MG1655 genome (NCBI RefSeq accession: NC_000913.3). Reads that had assorted primary hits or low mapping affection were discarded. Abeyant PCR duplicates were removed by application abandoned one brace of reads with the accomplished mapping affection back assorted apprehend pairs were mapped to identical alien coordinates (Picard Tool MarkDuplicates). BedGraph files that abode the concrete genomic advantage (taking into annual the unsequenced allotment amid apprehend pairs) in anniversary 2000-bp bin were generated from BAM files application deepTools (85). For ChIP-seq data, the apprehend counts in the bedGraph files were normalized to the average coverage. For whole-genome sequencing data, the apprehend counts in anniversary bin were aboriginal normalized adjoin absolute apprehend counts, and again the log2 ratios of DSB and no-DSB samples were calculated. Plots were generated by R software. Genomic regions that accommodate ribosomal RNA gene clusters had actual few abnormally mapped reads and were appropriately alone from the plots. All sequencing abstracts are accessible in the European Nucleotide Archive (ENA) beneath abstraction accretion no. PRJEB14145.

Replication origins were counted to appraisal the cardinal of forks per corpuscle by application rifampicin runoff analyses, as declared by Joshi et al. (56) and Bremmer and Dennis (86). Saturated brief cultures were adulterated 1:1000 or 1:750 in LBH glucose or M9 glucose medium, respectively, absolute doxycycline (100 ng/ml). Afterwards extensive OD600 (optical body at 600 nm) = 0.1, rifampicin and cephalexin were added to the culture, which was accustomed to complete advancing circuit of archetype (runoff). Beef were anchored and decrepit with 4′,6-diamidino-2-phenylindole and again analyzed on a BD Biosciences LSRFortessa cytometer. Three absolute abstracts were performed and after-effects were averaged.

The phage λ applique appraisal was declared by Shee et al. (39), except for consecration of Gam protein by alive temperature. The announcement of Gam was controlled by phage λ PR promoter, abutting to the λcIts857 gene, encoding a temperature-sensitive CI transcriptional repressor protein that represses λ PR at ≤33°C and allows archetype from λ PR at ≥37°C (39). Saturated tryptone borsch (TB) cultures of E. coli developed at 30°C were adulterated 1:10 in beginning TB with 0.2% maltose, 5 mM MgSO4, thymine (10 μg/ml), and vitamin B1 (10 μg/ml) and developed for bisected an hour at 30°C afore alive temperature to 37°C to abet accumulation of Mu Gam (or were developed at 30°C continuously). Afterwards 2.5 hours of induction, an according aggregate of 10 mM tris and 10 mM Mg (TM) absorber (pH 7.5) was added, and beef were alloyed with an adapted aggregate of λred gam Chi0 suspension, adsorbed afterwards afraid for 10 min at allowance temperature, and again argent with the accretion of 2.5 ml of aqueous bendable BBL agar [1% BBL trypticase peptone, 0.1 M NaCl, 0.7% agar (pH 7.5)] assimilate BBL plates (same average caked with 1% agar). The plates were incubated brief at 37°C or for 24 hours at 30°C afore celebratory the admeasurement of plaques (fig. S13). The accumulation of Mu Gam allows ample applique accretion by λred gam (39) [reviewed by Smith (87)].

Cancer accommodating RNA sequencing alternation analyses were performed application Spearman’s rank alternation on abstracts from cBioPortal (88, 89). RNA levels of specific genes in accommodating samples for the eight best accepted blight types—breast, lung, astute myeloid leukemia, colon, kidney, thyroid, bladder, and prostate—were compared with those in the cBioPortal advertence citizenry to accomplish Z scores, and the Z array were compared for anniversary gene of absorption adjoin the analogously generated Z array of accretion gene (for example, BLM mRNA adjoin RAD51 RNA; credible in Fig. 7A). The advertence populations for anniversary gene/cancer are all blight samples that are diploid for the gene in catechism (by absence for mRNA). The alternate amount indicates the cardinal of SDs abroad from the beggarly of announcement in the advertence citizenry (Z score). This admeasurement is advantageous for free whether a gene is up- or down-regulated about to all added bump samples. As abundant in table S1, the cardinal of accommodating sample abstracts sets per blight blazon ranged from 129 to 1100 (table S1). Spearman’s alternation accessory was computed to appraise the accord amid mRNA levels amid genes of interest.

Wet-bench experiments: All were performed at atomic three times independently, and a two-tailed unpaired t appraisal was acclimated to actuate cogent differences unless contrarily specified. Error bedfast represent 1 SEM except area contrarily indicated. Pearson’s alternation accessory was computed to appraise the accord amid RDG foci/cell and altered doses of γ-irradiation. Changepoint analyses were acclimated to ascertain peaks in ChIP-seq abstracts by audition the articulation credibility of curves and were performed application the “changepoint” R amalgamation (90).

Acknowledgments: We acknowledge J. D. Wang and S. J. Sandler for accouterment E. coli strains with a aberrant recA-gfp fusion; C. Shee for accessible discussion; F. Gu for cytometry help; M. A. Mancini, F. Stossi, R. D. Danderkar for advice with diminutive imaging; I. Golding and L. McLane for advice with microfluidics; and J. Gibson, S. G. Kozmin, K. M. Miller, F. W. Stahl, and three bearding reviewers for convalescent the manuscript. Funding: This assignment was accomplished with abutment from NIH Director’s Pioneer Award DP1-CA174424 (to S.M.R.) and developed via a allowance from the W.M. Keck Foundation (to S.M.R.). The assignment was additionally accurate by the National Aeronautics and Space Administration (NASA) through the NASA Astrobiology Institute beneath Cooperative Acceding No. NNA13AA91A issued through the Science Mission Directorate (to P.J.H.); Blight Blockage and Research Institute of Texas (CPRIT) admission RP140553 (to S.M.R.); CPRIT Baylor College of Medicine (BCM) Comprehensive Blight Training Program Postdoctoral Fellowship admission RP160283 (to D.M.F.); NIH R01 grants CA190635 (to L.L.), GM102679 (to D.B.), GM106373 (to P.J.H.), and GM88653 (to C.H.); NSF admission MCB1049925 (to M.J.); Robert F. Welch Foundation Award F-1274 (to M.J.); the BCM Cytometry and Corpuscle Sorting Amount with allotment from the NIH (P30-AI036211, P30-CA125123, and S10-RR024574); and the BCM Integrated Microscopy Amount with allotment from the NIH (HD007495, DK56338, and CA125123), CPRIT (RP150578), the Dan L Duncan Comprehensive Blight Center, and the John S. Dunn Gulf Coast Consortium for Chemical Genomics. Author contributions: S.M.R. conceived the study. J.X., L.-T.C., Q.M., L.L., D.B., P.J.H., C.H., M.J., and S.M.R. advised the study, analyzed the data, and wrote the paper. L.-T.C., J.X., Q.M., C.-H.M., J.A.H., H.-Y.L., D.M., J.P.P., D.M.F., H.M.H., M.R., R.B.N., and X.S. performed the experiments. Aggressive interests: The authors acknowledge that they accept no aggressive interests. Abstracts and abstracts availability: Sequencing abstracts are accessible in the ENA beneath abstraction accretion no. PRJEB14145. All abstracts bare to appraise the abstracts in the cardboard are present in the cardboard and/or the Supplementary Materials. Added abstracts accompanying to this cardboard may be requested from the authors.

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