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To delving the aboriginal accomplish of abrasion meitotic recombination, we activated END-seq16,17,18. In this method, a sequencing adapter is ligated to anniversary end of a DNA breach axial an agarose bung afterwards a accumulated of nucleases ExoVII and ExoT removes ssDNA overhangs. As a result, sequencing reads activate at the terminal end of physiological resection, connected in libraries of ssDNA-DSB junctions (Fig. 1b). These exonucleases can be acclimated to ascertain DSB termini that are either protein-bound or protein-free. For example, etoposide-induced DSBs, which are covalently absorbed to topoisomerase 2 (TOP2) via an animate armpit tyrosine at the 5′-termini, crave ExoVII to abolish covalently apprenticed TOP216, admitting ExoT can abandoned edgeless protein-free overhangs, connected in a ligatable DNA end18. Like TOP2, the topoisomerase-like protein SPO11 charcoal absorbed to DSB 5′-ends above-mentioned to absolution by MRE11-mediated nicking as abbreviate 20–40 bp oligonucleotides (SPO11 oligos)7,19 (Fig. 1a).



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We assayed spermatocytes from adolescent abrasion testes during the aboriginal beachcomber of semi-synchronous meiosis I. We anchored spermatocytes from 20 pooled, 12–14 dpp C57BL/6 J (B6) mice in agarose plugs and blunted meiotic ssDNA overhangs with ExoVII and ExoT afore ligating sequencing adapters (Fig. 1b). Approximately 5000 reproducible burst hotspots were alleged with a beginning of at atomic 2.5-fold accessory (Supplementary Fig. 1a, Supplementary Data 1). We overlapped these END-seq peaks with ahead appear B6 meiotic hotspots bent by SPO11-oligo sequencing and DMC1 single-strand DNA sequencing (SSDS)8,11,20. By beheld assay and allusive analysis, both END-seq breach breadth and aiguille acuteness overlapped with SPO11 oligos and SSDS, which accounted for 97% and 98% of END-seq peaks, appropriately (Fig. 2a, Supplementary Fig. 1b, c). END-seq accordingly provides a map of anon detected meiotic DSB resection in mammals.

a Representative genome browser profiles of meiotic hotspots for SPO11-oligo sequencing, DMC1 SSDS, and END-seq. Browser arbor scales are adapted amid techniques to appearance both hot and algid hotspots simultaneously. b Heatmaps of END-seq, SPO11-oligo, and SSDS ± 2.5 kb about hotspot centers (determined by SPO11-oligo summits), ordered by absolute apprehend calculation of END-seq, for top 5000 END-seq breaks. WT END-seq peaks are provided in Supplementary Data 1. c Schematic of END-seq breach pattern, consisting of (1) a axial aiguille at the SPO11 breach armpit (2) a read-less gap produced by MRE11-mediated short-range resection and minimum ambit of all-embracing resection, and (3) distal reads at the terminal ends of all-embracing resection. This arrangement is axiomatic at abandoned hotspots (middle, chr1:68488000–68491500) and back arresting from all hotspots is aggregated (bottom). Minimum resection lengths are affected by the absence of sequencing reads (blue accent region); beggarly and best all-embracing resection are affected by the boilerplate and best distal reads from the DSB, appropriately (red accent region). d END-seq axial aiguille and SPO11-oligo reads are ancillary as credible by aggregated arresting about SPO11-oligo summits (normalized to the aforementioned height). Both the primary SPO11 aiguille and adjoining accessory peaks are apparent. e Accumulated arresting allegory of murine meiotic END-seq about SPO11-oligo summits and aggrandize meiotic S1-seq about aggrandize hotspot centers. f Aggregated END-seq and H3K4me3 ChIP-seq arresting (normalized to the aforementioned height).



Using the 2.5-fold-enrichment criteria, END-seq peaks alleged a third of the absolute hotspots bent by SPO11-oligo seq or SSDS (Supplementary Fig. 1b, c). However, END-seq peaks that did not accommodated this blow were about associated with ahead mapped hotspots. For example, 66% of SPO11-oligo loci that were “END-seq negative” showed the aforementioned DSB apprehension arrangement back these END-seq reads were aggregated (Supplementary Fig. 1d, e). Moreover, these END-seq-negative SPO11-oligo sites appearance a cogent abridgement in SPO11-oligo reads (Supplementary Fig. 1f), advertence that these are the coldest meiotic hotspots. We accomplish that END-seq detects accident at all ~ 15,000 ahead mapped hotspots, yet the added frequently burst top 33% of hotspots crop the best able-bodied signal. Therefore, unless contrarily stated, the consecutive analyses were performed on the 5000 arch END-seq breaks.



END-seq captured a conspicuously compatible arrangement of accident at all sites that consisted of a able axial aiguille anon at the armpit of SPO11 acid with an accession of reads crabbed the cut armpit at a authentic ambit abroad (Fig. 2b, c). We adapt the axial aiguille to be the absolute apprehension of SPO11 accident while adjacent, distal reads reflect minimum and best resection endpoints in the citizenry of spermatocytes. Reads absolute the axial aiguille are absolutely ancillary with SPO11-oligo mapping and reflect a subset of breach aural the spermatocyte citizenry that has not yet appear covalently apprenticed SPO11 from the DSB (Fig. 2d). As discussed later, the axial aiguille in WT beef is abased on assurance of the cut chromosome with the uncut homolog, and accordingly represents unreleased SPO11 associated with recombination.

END-seq detects the terminal end of physiologic resection afterwards in vitro abatement of the 3′ overhang, with the aboriginal nucleotide sequenced agnate to the position of the ssDNA-DSB alliance (Figs. 2c, 1b)18. If bidirectional resection of DNA-bound SPO11 by short- and all-embracing resection machineries are absolutely accommodating as in yeast4,5,6, afresh the ssDNA-DSB alliance will consistently be aloft the best distal MRE11 nick and accordance to the terminal end of all-embracing resection (Figs. 2c, 1b). In this case, the breadth of short-range 3′−5′ MRE11 exonuclease action would not crop any sequencing reads as it does not accomplish apart of 5′−3′ resection (Figs. 2c, 1b). Indeed, at every hotspot, we empiric a read-less “gap” in resection, connected with the apprenticed coupling of resection admission (by MRE11) and 5′−3′ addendum by the all-embracing resection accouterment (Fig. 2b, c). The breadth of this gap reflects the minimum resection endpoints in the spermatocyte citizenry and corresponds to the best ambit from the DSB at which MRE11 nicks the fiber added any constant, minimum ambit that 5′−3′ exonucleases traverse. This arrangement is evocative of S1 nuclease apprehension of meiotic recombination in yeast6 (Fig. 2e). Thus, both SPO11 acid and its antecedent processing by accommodating resection mechanisms are awful conserved evolutionary appearance of meiotic recombination that bulk unicellular eukaryotes to mammals.

The gap admeasurement was acutely compatible at all hotspots, with beggarly best ambit of 647 nts (Supplementary Fig. 2a), and was abundantly belted to the two-nucleosome H3K4/K36me3 arresting surrounding SPO11 cut sites as bent by ChIP-seq (Fig. 2f, Supplementary Fig. 2b). Thus, minimum resection distances associate able-bodied with PRDM9-mediated methylated histone deposition10. Interestingly, the majority of CO and NCO boundaries in mice are additionally belted to this region, rarely extending aloft the 650 bp gap (Supplementary Fig. 2c)15,21. Because minimum resection activated able-bodied with methylated histones, we asked if all-embracing resection endpoints were analogously able-bodied positioned. Amid two carbon END-seq samples, we begin aerial alternation in the arrangement of resection endpoints amid the hotter hotspots detected with alternate subpeaks credible at a beggarly ambit of 210 nucleotides (Supplementary Fig. 2d, e). This suggests that nucleosome ascendancy far from the breach armpit may access all-embracing resection endpoints.

To actuate the acuteness of the method, we compared 20 affiliated adolescent mice to a library fabricated from a audible 12 dpp mouse. Both samples alleged ~ 5000 peaks, with 77–89% accumulated breach with awful activated (r = 0.98) END-seq intensities (Fig. 3a). Importantly, the breach arrangement at abandoned hotspots was absolutely retained in testes from a audible abrasion (Fig. 3b). To analyze signal-to-noise arrangement (S/N) in the two libraries, we followed ENCODE’s appraisal of atom of reads in peaks (FRiP) and cross-correlation profiles (CCPs) for ChIP-seq abstracts sets22,23. FRiP and CCP ethics for both libraries exceeded ENCODE’s belief for signal-to-noise ratios (Fig. 3c, d). We accomplish that END-seq can accurately appraise abandoned meiotic DSB locations and processing with appreciably little biological material. This aerial acuteness bypasses the limitations of added hotspot mapping methods that crave either abstract quantities of mice (SPO11-oligo seq) or the availability of species-specific, high-quality antibodies (DMC1 SSDS).

a Larboard panel: alternation (Pearson’s r) amid END-seq RPKM from 20 mice against RPKM from one 12 dpp mouse ± 3 kb about SPO11 summits. Appropriate panel: Venn diagram overlap of alleged peaks amid 20 mice and one mouse. P value < 2.2e-16, Fisher’s exact test. b Larboard panel: Full END-seq breach arrangement in aggregated arresting from one mouse, centered about SPO11 oligos. Appropriate panel: heatmap of END-seq arresting from one abrasion in a ± 3 kb window about SPO11 oligos, ordered by absolute apprehend count. c FRiP ethics for 20 mice against one mouse. Recommended ENCODE bulk denoted by dotted red line. d Cross-correlation artifice profiles for 20 mice and one mouse. The artifice shows Pearson cross-correlations (CCs, y axis) of apprehend intensities amid the added fiber and the bare strand, afterwards animate bare fiber (x axis). One aiguille corresponds to apprehend breadth (CCread, dejected birr line) and the added one corresponds to the fragment breadth (CCfrag, red birr line). Normalized fiber accessory (NSC) is CCfrag disconnected by basal CC bulk (CCmin) and about fiber accessory (RSC) is the arrangement of CCfrag-CCmin disconnected by CCread-CCmin. Higher NSC and RSC ethics beggarly added enrichment. ENCODE’s advocacy for ChIP-seq: NSC ≥ 1.05 and RSC ≥ 0.8.

ATM is anticipation to abnormally adapt SPO11 cutting, and in its absence, hotspot accident and SPO11 oligos accept been apparent to clearly access in mice7. This ultimately after-effects in aboriginal meiotic arrest and apoptosis of Atm–/– spermatocytes that backpack an balance of unrepaired DSBs7. To added validate this archetypal application direct, genome-wide detection, we performed END-seq on Atm–/– spermatocytes that were backcrossed 11 times to the B6 background. Indeed, Atm–/– END-seq detected 99% of WT END-seq breach while calling ~ 6300 more peaks (Supplementary Fig. 3a, Supplementary Data 2). All Atm–/– breach overlapped bigger with SPO11 oligos and SSDS sites and showed amplified arresting over WT at anemic SSDS hotspots (Supplementary Fig. 3b–d). These abstracts affirm antecedent letters that colder hotspots become preferentially hotter in the absence of ATM attributable to a accident of abrogating acknowledgment of SPO11 acid (Supplementary Fig. 3e)7,8. To quantify this increase, we added a spike-in normalization ascendancy to END-seq plugs, consisting of a G1-arrested Ableson-transformed murine pre-B corpuscle band (Lig4–/–) accustomed a audible zinc-finger-induced DSB at the TCRβ enhancer16, which was alloyed in at a 2% affluence with accumulated testiscular cells. Afterwards normalizing END-seq reads at all ~ 15,000 SPO11-oligo hotspots application the arresting at the TCRβ enhancer, we begin that Atm–/– beef anchorage a 4.5-fold access in absolute breach over WT. As discussed later, added SPO11 double-cutting aural the aforementioned hotspot may annual for the 10-fold access in SPO11 oligos appear in Atm–/– spermatocytes;7 yet END-seq would ascertain double-cutting as a audible event, acquiescent a 4.5-fold access in DSBs.

Unexpectedly we begin that 7–16% (700–1800) of breach sites were not accumulated in WT SSDS or SPO11-oligo maps, appropriately (Supplementary Fig. 3b, c). Amid these were several hundred apostle breaks, about associated as “default” hotspots in bacilli abnormal PRDM9 (Supplementary Fig. 3f). Indeed, these breach locations are amid the hottest Prdm9–/– sites as bent by SSDS (Supplementary Fig. 3g)11,24. Thus, in the absence of ATM, colder hotspots and absence hotspots become added broken. This is connected with antecedent observations that ATM-null spermatocytes accept added SPO11-oligo mapping at PRDM9-independent hotspots14.

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Long-range resection endpoints showed greater aberration (1–3 kb) about to the minimum resection gap arena (0.4–1 kb) (Supplementary Fig. 2a). Antecedent studies estimated absolute beastly meiotic resection lengths based on the admeasurement of DMC1 apprenticed to ssDNA overhangs as abstinent by DMC1 SSDS8. Strikingly, we begin that best resection endpoints continued decidedly added at all hotspots than DMC1-bound ssDNA (Fig. 4a, b). ssDNA active by DMC1 ranged from 800 to 2700 nts, admitting best all-embracing resection lengths (defined in Fig. 2c) bent by END-seq were 1.2–1.6-fold greater (Fig. 4b, c). These abstracts announce that DMC1 binds to abandoned a allocation of the accessible ssDNA and underestimates the absolute admeasurement of meiotic resection. One abeyant account is that audible advance of ssDNA can be co-occupied by DMC1 and RAD51 filaments with DMC1 loaded added proximally to the breach armpit than RAD5125. Although unlikely, this aberration may additionally be due to abstruse limitations in SSDS library alertness that relies on microhomology-mediated ambit formations artlessly present in ssDNA tracks26.

a Top ( ) and basal (–) fiber distributions of END-seq and SSDS appearance added resection apprehension by END-seq (left, arresting normalized to the aforementioned height) that is axiomatic at abandoned hotspots (right). b Boxplot of END-seq vs SSDS best resection per hotspot. ****p < 1e-10; t test. c Alternation (Pearson’s r) of best resection endpoints detected by END-seq and SSDS. d Accumulated artifice of END-seq arresting in WT and Exo1–/– spermatocytes (signal normalized to the aforementioned height) about SPO11 oligos. e Boxplots of beggarly resection (as authentic in Fig. 2c) amid WT, Exo1–/–, 53bp1–/–, Brca1Δ1153bp1–/–, Brca1Δ1153bp1S25A and Brca1Δ11p53 /– at top 250 hotspots. *p < 0.01; ****p < 1e-10; t assay with mu = 10 (mu is estimated as accepted aberration of WT replicates). f Boxplots of best resection endpoints amid WT, Dmc1–/–, and Hop2–/– at top 5000 hotspots. *p < 0.01; ****p < 1e-10; t assay with mu = 43 (mu is estimated as accepted aberration of WT replicates). g, h Histogram distributions comparing either WT and Dmc1–/–g or WT and Atm–/–h END-seq minimum and best resection endpoints at top 5000 hotspots. Beggarly ethics (bp) are listed.

Because the resection arrangement in abrasion mirrored so carefully that empiric in aggrandize (Fig. 2e)6, we capital to apperceive whether they activated the aforementioned all-embracing end-processing machineries. Exo1-deficient aggrandize display absolute accident of 5′−3′ all-embracing resection3. In contrast, EXO1 and DNA2 act redundantly in aggrandize abounding and beastly actual beef to arbitrate end resection27,28,29. Surprisingly, END-seq assay of adolescent Exo1–/– spermatocytes appear that all-embracing resection was abundantly complete (Fig. 4d). Back averaged genome-wide, we begin a small, admitting significant, abridgement in all-embracing resection ambit in Exo1–/– beef (median resection tract: WT: 1,129 nts vs. Exo1–/–: 996 nts (Fig. 4e). We accordingly accomplish that compared with yeast, beastly meiosis has acquired added mechanisms to accomplish all-encompassing 3′ overhangs, conceivably through appliance of bombastic DNA2 exonuclease activity27,28,29.

In actual interphase cells, 53BP1 has been apparent to arrest all-embracing resection of DSBs30. One principal role of BRCA1 is to annul 53BP1’s block to resection in S phase, possibly by excluding it from chromatin adjoining to DNA accident sites31. In addition, BRCA1 acts post-resection to bulk the RAD51 recombinase assimilate 3′ ssDNA32. Despite these all-encompassing studies demonstrating their accent in actual cells, the roles of 53BP1 and BRCA1 in acclimation meiotic resection are unknown. We accordingly performed END-seq on 53bp1–/– and BRCA1-deficient spermatocytes and abstinent all-embracing resection lengths. In arresting adverse to 53BP1-deficient actual beef in which DSB resection is consistently and acutely increased18,30,33, we begin that resection lengths were commensurable at all meiotic hotspots in 53bp1–/– and WT spermatocytes (Fig. 4e).

We afresh assayed Brca1Δ11p53 /– mice, which display accepted defects in BRCA1 function, yet are animate attributable to fractional p53 apoptotic suppression34. Contrary to our expectations30,33, Brca1Δ11p53 /– spermatocytes showed a balmy access in DSB resection about to WT controls (Fig. 4e). Moreover, Brca1Δ1153bp1–/– and Brca1Δ1153bp1S25A spermatocytes33 apparent a agnate access in resection as Brca1Δ11p53 /− spermatocytes (Fig. 4e). Based on these findings, we accomplish that in adverse to their categorical afraid relationship during DSB processing in interphase cells, 53BP1 does not arrest resection while BRCA1 does not advance resection during meiotic recombination. As discussed below, one acumen why end resection increases (rather than decreases) in BRCA1-deficient beef may be attributable to inefficient loading of the recombinase. Connected with this, Brca1Δ11p53 /– spermatocytes accept bargain RAD51 and DMC1 focus counts35.

In beginning yeast, beef abnormal DMC1 appearance hyper-resection, conceivably attributable to a abrogating acknowledgment on resection advised by recombinase loading4,6. Given our after-effects that BRCA1-deficient spermatocytes apparent added resection (Fig. 4e) associated with abnormal RAD51/DMC1 foci35, we accepted that RAD51 or DMC1 loading assimilate ssDNA ability absolute all-embracing resection. Connected with this, Dmc1–/– spermatocytes showed essentially added resection about to WT at all hotspots (Fig. 4f, g and Supplementary Fig. 4a). Minimum resection lengths additionally added in Dmc1–/– beef by ~ 400 nts about to WT (Fig. 4g and Supplementary Fig. 4a).

HOP2 protein is appropriate for able homolog band afterwards loading of DMC1/RAD51 recombinases36. In Hop2−/− spermatocytes, resection lengths were agnate to WT (Fig. 4f). Thus, recombinase loading post-resection, absolute of fiber invasion, banned both the minimum and best breadth that the all-embracing resection accouterment processes DSBs. DMC1/RAD51 loading assimilate ssDNA ability absolute resection by preventing the re-initiation of exonucleases on already resected ssDNA.

ATM abnormally regulates DSB consecration by SPO117. Despite the actuality that Atm–/–spermatocytes anchorage a 10-fold access in SPO11 oligos7, RAD51/DMC1 foci anatomy at agnate levels as in WT, admitting RPA foci counts are added in aberrant cells37. One accessible acumen why the cardinal of foci does not associate with the ample access in DSBs could be that recombinase akin or action is attached fiber formation. In this scenario, back balance DSBs are generated in an ATM-null background, the basin of RAD51 and/or DMC1 is bereft to bulk assimilate all ssDNA regions. In accordance with this hypothesis, the beggarly best resection lengths added about bifold in Atm–/– spermatocytes about to WT (WT: 1845 nts. vs. ATM: 3351 nts; Fig. 4h). Moreover, in ATM knockouts, resection lengths were decidedly greater and added broadly broadcast than alike DMC1 knockouts (Fig. 4g, h), conceivably attributable to accumulated absence in RAD51 and DMC1 loading assimilate ssDNA37. In accordance with the abstraction that added DSB numbers rather than absence in ATM signaling contributes to the hyper-resection phenotype in the ATM-null background, S1-seq assay of Atm–/–Spo11 /– spermatocytes, in which SPO11-oligo complexes are bargain by half7, appear accustomed resection lengths38. Together these abstracts abutment the abstraction that the availability of recombinases to anatomy filaments is attached and bereft back there is boundless SPO11 cutting.

In accession to the hyper-resection empiric at a subset of breaks, audible boundaries amid the short- and all-embracing resection about abolished in Atm–/– spermatocytes. This resulted in reads mapping aural the gap arena (Fig. 5a, b), in adverse to WT that consistently generates ssDNA-DSB junctions aloft the best distal MRE11 nick. The decreased minimum resection could announce that ATM regulates the deeply accompanying activities of short-range and all-embracing resection (Fig. 5c).

a Accumulated plots of END-seq arresting in WT vs Atm–/– at top 5000 WT breach about SPO11-oligo summits. Signals are normalized to the aforementioned acme for DSB arrangement clarity. b Heatmaps of WT and Atm–/– END-seq ± 5 kb about SPO11-oligo summits for top 5000 WT hotspots. Signals are normalized to spike-in ascendancy to appearance added Atm–/– breach acuteness per hotspot. c Illustrations of ATM’s assorted roles in akin resection that gives all-embracing amalgamate END-seq arrangement in a. Left: MRE11 is recruited yet not activated in a subset of cells, connected in reads anon at SPO11 DSB aural the population. Middle: MRE11 is activated in addition subset of cells, yet all-embracing resection is not abundantly initiated, connected in reads from abandoned MRE11 nicking, and conceivably 3′−5′ exonuclease activity, crabbed the DSB in regions that are read-less in WT. Right: resection is appropriately accomplished in addition subset of cells, yet all-embracing resection campaign decidedly added than in WT. d Aggregated END-seq arresting and MRE11 ChIP-seq RPM in WT (left) and Atm–/– (right). To adequately analyze ChIP-seq arresting amid WT and Atm–/–, MRE11 calibration is proportional to spike-in normalized END-seq RPM for anniversary genotype. Abandoned hotspot examples (chr12:34,592,264-34,598,265) are apparent below. Note that decreased MRE11 advantage is empiric aural NDR of Atm–/–. e Accumulated artifice overlapping WT END-seq and Atm–/– MRE11 ChIP-seq, normalized to the aforementioned height. MRE11 shows arresting localization to the WT END-seq read-less gap.

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If some DSBs abide unresected in ATM-deficient spermatocytes, we would adumbrate that MRE11 would accrue at these chapped DNA ends. Connected with this hypothesis, ChIP-seq for MRE11 appear cogent chromatin bounden at all hotspots in Atm–/– beef (Fig. 5d, right, Supplementary Fig. 4b), which activated with END-seq acuteness (Supplementary Fig. 4c). This likely reflects a role for ATM in announcement accustomed MRE11 resection admission as appropriate for Tel1 in yeast6. In contrast, we detected no arresting in analogously burst regions in WT beef (Fig. 5d, left), apocalyptic of completed resection. Strikingly, the MRE11 arresting in ATM-null beef was absolute aural the WT read-less gap (Fig. 5d, e, Supplementary Fig. 4b). This lends added abutment to the abstraction that the gap reflects MRE11-dependent end-processing. Interestingly, MRE11 bounden abundantly beneath in the baby axial H3K4me3/K36me3 nucleosome-depleted breadth (NDR) (Fig. 5d, e and Supplementary Fig. 4b). The NDR in Atm–/– beef may be depleted of DNA attributable to SPO11 double-cutting (see below).

At all hotspots, END-seq detected a compatible accession of reads accumbent to the centermost of the DSB. This axial aiguille was ancillary with mapped SPO11 oligos, accepting a amplitude (400 bp) agnate to SPO11-oligo seq hotspots (300–400 bp) and was belted to the NDR of H3K4/K36me3 (Fig. 2d,f and Supplementary Fig. 2b)8. Moreover, alike low-level accessory oligos that are adjoining to the axial SPO11 hotspot peak8 were apparent by END-seq (Fig. 2d). This appropriate that the axial aiguille represented a atom of the absolute DSBs in the spermatocyte citizenry in which SPO11 is not yet released, thereby highlighting the adverse of cellular DSB processing.

If 5′ covalently apprenticed SPO11 DSBs existed at the time of END-seq processing, afresh the proteinase K assimilation would leave abaft a two-bp 5′ overhang with a phosphotyrosyl band that requires ExoVII assimilation to absolutely edgeless the end, akin to our studies on TOP2 breach complexes (TOP2cc)16. To assay this hypothesis, we performed END-seq with ExoT abatement only, acceptance adapter articulation abandoned to protein-free DNA ends while excluding ends with protein adducts, such as any actual trapped SPO11 breach complexes (SPO11cc). Strikingly, ExoT detected abandoned absolutely resected DNA ends with a absolute absence of axial arresting at all hotspots (Fig. 6a and Supplementary Fig. 4d). These abstracts announce that the axial peak, which represents ~ 11% of the absolute DSB arresting and is present all hotspots, reflects SPO11 covalently apprenticed to its breach site.

a END-seq processing with ExoT abatement abandoned shows absolute absence of SPO11 axial aiguille back arresting is aggregated about SPO11-oligo summits (left). Heatmap of ExoT abandoned arresting ± 3 kb about hotspot centers, ordered by absolute apprehend calculation of END-seq (right). b Pretreatment with antiseptic animal MRN   CtIP reduces ExoVII   ExoT axial aiguille apprehension (red) over no pretreatment (NT, black) and depends on the attendance of CtIP (blue). Both MRN and MRN   CtIP reactions were agitated out in the attendance of manganese. One 12 dpp abrasion acclimated per condition. c END-seq with ExoT abatement abandoned additionally shows no axial aiguille in Atm–/– beef (black line). Pretreatment with antiseptic animal TDP2 afore ExoT evolution recovers SPO11 signal, advertence affluence of unresected, bona fide SPO11 breach complexes (red line). Accumulated artifice (left, normalized to aforementioned resection height) and heatmap (right) ±  3 kb about SPO11-oligo summits. d END-seq accumulated artifice of WT ExoT abatement with (red line) and afterwards (black line) TDP2 pretreatment shows abandoned accessory accretion of axial aiguille signal.

To validate that the ExoVII-dependent axial aiguille was attributable to SPO11 apprenticed to the breach and not some added anatomy of occlusion, we speculated that it ability be accessible to abolish SPO11 tyrosyl-linked DNA through evolution with antiseptic animal MRE11/RAD50/NBS1 (MRN) and CtIP above-mentioned to abatement with ExoVII   ExoT, artful the in vivo processing of meiotic DSBs. Indeed, we empiric a affecting accident of axial arresting by preincubation with MRN and CtIP (Fig. 6b)39. Evolution with MRN abandoned (prior to ExoVII   ExoT) or MRN and CtIP in the absence of manganese did not calmly abolish the axial aiguille (Fig. 6b and Supplementary Fig. 4e) connected with the award that CtIP and manganese is appropriate for the in vitro MRE11 endonuclease processing of protein-bound DSBs40,41. Thus, antiseptic MRN   CtIP recognizes and removes the actual SPO11 phosphotyrosyl bonds associated with the axial signal. These abstracts accordingly abutment the abstraction that a atom of SPO11 charcoal physiologically apprenticed to a subset of breach (~ 11%) at about all hotspots afterwards cutting.

Because Tel1 regulates MRE11-initiated resection, there is a apparent access in unresected DSBs in Tel1-deficient yeast6. If ATM functions analogously to Tel1, afresh we would apprehend an accession of unresected SPO11cc, able-bodied aloft WT levels, at the centermost of the hotspots. The abounding MRE11 ChIP-seq arresting accurately in Atm–/– beef (Fig. 5c, d), apocalyptic of abridged processing, would additionally adumbrate a all-inclusive access in SPO11cc in the mutant.

We accordingly approved to adapt END-seq to accurately delving for SPO11cc. We accepted that preincubation with antiseptic tyrosyl-DNA phosphodiesterase 2 (TDP2)42 would abolish the actual phosphotyrosyl adduct afterwards proteinase K treatment, breeding a two-nucleotide, protein-free 5′ overhang that ExoT could readily edgeless for adapter ligation. Although ExoT abandoned detected no axial aiguille in ATM-null cells, agnate to WT (Fig. 6c), TDP2   ExoT END-seq captured an astonishingly able-bodied axial arresting (Fig. 6c). Moreover, this aiguille was acerb detected at all hotspots (Fig. 6c, right). SPO11cc apprehension by TDP2   ExoT far exceeded the ability of apprehension with ExoVII   ExoT in Atm–/– spermatocytes both at autosomes and at the non-PAR X chromosome (Supplementary Fig. 4f, g), best acceptable absorption the biochemical alternative for TDP2 over ExoVII to abolish the phosphotyrosyl bonds and acquiesce for adapter ligation42. These abstracts announce that ATM-deficient spermatocytes accrue unresected SPO11cc, agnate to aggrandize abnormal Tel16.

In WT cells, the ExoVII   ExoT axial signal, which is additionally SPO11-bound, represents 11% of the absolute DSB fraction. Yet, in adverse to Atm–/–, accumulated of TDP2 and ExoT abandoned recovered a baby atom (16%) of the ExoVII   ExoT axial arresting begin in WT beef (Fig. 6d), agnate to 0.5–2% of the absolute DSB signal. Given the aerial ability of TDP2   ExoT in ATM-null beef for apprehension of SPO11cc, it was initially cryptic why the WT TDP2   ExoT axial arresting was so low. We speculated that in WT cells, the axial arresting ability not artlessly reflect unresected SPO11cc, as empiric in Atm–/– spermatocytes. Rather, there ability be an added anatomy at the breach armpit associated with SPO11-bound DNA in WT beef that somehow prevented acceptance by TDP2.

Our aboriginal clue to compassionate the audible attributes of SPO11-bound DNA in WT vs. Atm–/– beef was the ascertainment that it was missing from all hotspots in Dmc1–/– spermatocytes (Fig. 7a). This prompted us to ask whether the axial aiguille associated with DNA-bound SPO11 was additionally abased on ssDNA fiber aggression into the akin partner. Remarkably, we begin a accident of axial arresting at all non-PAR X chromosome hotspots (Fig. 7b), which adjustment from the sister chromatid as the X chromosome has no homolog in males. The balance axial arresting resembled the TDP2   ExoT arresting in WT beef (Fig. 6d) and accordingly ability artlessly reflect the bulk of unresected SPO11 artlessly present on all chromosomes. In arresting contrast, the axial arresting associated with autosomes and the non-PAR X chromosome was about identical in Atm–/– spermatocytes, afresh acknowledging that the axial arresting is abundantly SPO11cc in the absence of ATM (Supplementary Fig. 4h–i). Finally, we begin that the axial arresting was absent in Hop2−/− spermatocytes (Fig. 7c), which absolutely bulk recombinases but abort to appoint the akin template36.

a Aggregated END-seq arresting about SPO11 oligos, with hotspot example, assuming absence of SPO11 axial aiguille in Dmc1–/–. b Abridgement in axial arresting at non-PAR X chromosome hotspots; aggregated arresting on ChrX compared with all autosomes. c Aggregated END-seq arresting about SPO11 oligos assuming absence of SPO11 axial aiguille in Hop2–/–. d Aggregated END-seq arresting of B6 (centered on B6 SPO11 oligos) against B6xCAST amalgam (centered on amalgam SSDS hotspot centers). e Prdm9–/– END-seq arresting aggregated about absence SSDS hotspot centers at ~ 200 sites with atomic overlap in SSDS top and basal strands. WT END-seq is centered about WT SPO11 oligos at PRDM9-dependent hotspots. f Prdm9–/– SSDS and END-seq advance at a audible absence hotspot (Yaf2 gene) with basal SSDS top and basal fiber overlap. Capital SPO11 breach armpit (red dotted line) is accepted from SSDS pattern. Accumulated plots in all panels are normalized to the aforementioned acme for beheld comparison.

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The assay of amalgam abrasion strains with altered PRDM9 alleles has appear that the amount of aberration in PRDM9 binding—that is, whether PRDM9 binds unequally to both homologs—predicts added asynapsis and amalgam infertility13,14. Back PRDM9 fails to bind the ceaseless akin chromatid, there is a astringent abridgement in both CO and NCO events14,15. Indeed, it has been appropriate that agnate to the X chromosome, agee hotspots ability be repaired from the sister chromatid15. We accordingly performed END-seq on spermatocytes acquired from adolescent B6xCAST hybrids with aberrant genomes. Strikingly, the axial aiguille was bargain to 2–3% of the absolute DSB arresting in B6xCAST compared with the 11% empiric in B6 (Fig. 7d) and abundant lower than in CAST abandoned (Supplementary Fig. 4j). Altogether, these allegation abutment the abstraction that the axial arresting detected by ExoVII ExoT in WT spermacotyes is associated with DNA-bound SPO11, and is abased on the amount of akin chromosome engagement.

In mice abnormal PRDM9, DSBs action at H3K4me3 sites mainly associated with promoters9,11,12. However, these DSBs are not repaired calmly as COs connected in meiotic arrest11. To assay how PRDM9 absence impacts homolog assurance and resection, we performed END-seq on Prdm9–/– spermatocytes. As SPO11 can accomplish assorted breach aural the H3K4me3 sites at promoters, we focused our analyses on Prdm9–/– SSDS sites that apparent the atomic overlap amid top and basal fiber reads, i.e., hotspots that are best acceptable to accept one capital SPO11 cut armpit aural the apostle (Supplementary Fig. 5a). Such SSDS hotspots showed apprenticed arresting at the center, in band with these sites accepting a able alternative for a audible SPO11 cut armpit (Supplementary Fig. 5a, b). Strikingly, END-seq assay of Prdm9–/– spermatocytes appear a absolute absence of the axial aiguille at hotspots with the atomic SSDS fiber overlap, advised either on accumulated or alone in the genome browser, admitting short- and all-embracing resection appeared to be almost complete (Fig. 7e, f and Supplementary Fig. 5c, d). The absence of axial arresting is connected with the abstraction that PRDM9 promotes homolog engagement, which in about-face facilitates COs. Alternatively, adjournment in homolog assurance ability acquiesce added time for processing DNA-bound SPO11, connected in accident of SPO11-bound DNA.

At a meiotic DSB that has been absolutely resected on both abandon and SPO11 absolutely released, adapters ligated to the appropriate end of the breach will adjust to the top ( ) DNA strand, admitting adapters ligated to the larboard end will adjust to the basal (–) fiber (Fig. 2c, Fig. 8a, left). Back reads from all hotspots are aggregated, the distal resection arresting about SPO11 cuts apparent this “correct” polarity for the top and basal strands (Fig. 8a, left). However, a abutting assay of the WT END-seq reads associated with the axial arresting accidentally appear a “wrong” polarity, in which top fiber reads accumbent hardly to the larboard aural the NDR and basal fiber reads accumbent hardly to the appropriate (Fig. 8a, right). As SPO11 generates a DSB with abandoned 2 nt overhangs, if the axial arresting were alone a accumulating of unresected SPO11cc, afresh accumulation the top and basal fiber DSB endpoints should appearance no breach amid them. However, we empiric a top and basal fiber about-face of ~ 60 nts in the “wrong” acclimatization (Fig. 8a, right). Detecting a cogent changeabout in the accepted polarity adumbrated that the axial aiguille was not artlessly SPO11cc, which should accomplish a approved DSB arrangement (see Atm–/– below). This appropriate that although SPO11 charcoal apprenticed to a atom DSBs, there is some affectionate of aberration associated with DNA-bound SPO11 that afflicted END-seq apprehension compared with back SPO11 is appear and both DNA ends are absolutely resected (Fig. 8a).

a At absolutely candy and resected SPO11 DSBs, END-seq top and basal fiber reads display a “correct” polarity to the appropriate and larboard of the DSB, appropriately (left). WT reads at the centermost of hotspots appearance a fiber polarity that is antipodal from what is accepted (right, zoomed in at NDR). b PRDM9 as a barrier to SPO11 processing that after-effects in a SPO11-bound recombination boilerplate (SPO11-RI) structure. In WT, SPO11 acid to one ancillary of chromatin-bound PRDM9 aural the NDR may block MRE11 action on one ancillary of the break, abrogation SPO11 covalently apprenticed to a abbreviate amplitude of dsDNA, capping the DMC1-loaded ssDNA that extends ~ 1 kb from the NDR. SPO11-RI is sequenced starting from the aboriginal SPO11-bound nucleotide area an adapter is ligated. SPO11 that cut larboard of PRDM9 would aftereffect in top fiber reads adjustment to the larboard of the PRDM9 burden and basal fiber reads from absolutely resected ssDNA adjustment at a ambit abroad from the breach site. c Atm–/– END-seq axial reads accept actual afar fiber polarity aural the NDR. d Unresected SPO11 double-cutting aural the aforementioned hotspot in Atm–/– beef would appearance the actual polarity of top and basal strands afterwards adapter articulation to SPO11-bound DSBs and ~ 50 bp separation, as empiric in c. Decreased MRE11 action at these breach would aftereffect in the absolute sequencing of SPO11 breach complexes aural the NDR rather than SPO11-RI in WT.

One abeyant apparatus that could accord to the amiss polarity is agee processing of SPO11. That is, if the two DNA ends apprenticed by SPO11 are candy at altered efficiencies by MRE11, one end ability be clumsily processed, abrogation SPO11 covalently apprenticed to its cut site, admitting the added end is candy to achievement and SPO11-oligo released. In this scenario, abandoned the clumsily candy SPO11-bound end would accord to axial aiguille signal. The absolutely candy end (Fig. 8b, top, larboard end of the DSB) would aftereffect in the absolution of the SPO11-oligo that would accomplish a protein-free 3′ overhang. This (SPO11-free) DNA end would in about-face be blunted by END-seq and sequencing reads would be detected aural the distal, all-embracing resection peaks (Fig. 8b, bottom, larboard end of the DSB). In contrast, the added ancillary of the DSB would be clumsily resected by MRE11 and absorb SPO11 covalently apprenticed to a two-nucleotide, 5′ overhang (Fig. 8b, top, appropriate end of the DSB). END-seq apprehension (with ExoVII   ExoT) would afresh abolish SPO11 and arrangement the actual dsDNA, with the aboriginal nucleotide sequenced actuality the SPO11 breach armpit itself (Fig. 8b, bottom, appropriate end of the DSB). This would aftereffect in top and basal fiber axial aiguille reads with antipodal polarity aural the NDR, as SPO11 breach to the larboard of the NDR centermost would accord top fiber reads adjustment larboard of center, and SPO11 breach to the appropriate would accord basal fiber reads adjustment appropriate of centermost (Fig. 8b). In a citizenry of spermatoctyes in which both contest action amid the cells, END-seq would ascertain an all-embracing aggregated signal of resection reads with actual polarity and axial reads with antipodal polarity (Fig. 8b, bottom).

How could such agee MRE11-mediated processing arise? Best SPO11-oligo-sequencing reads array in the centermost of the nucleosome-free depleted arena area PRDM9 is additionally bound, suggesting that PRDM9 does not block SPO11 access8,43. Rather, we brainstorm that DNA-bound PRDM9 may adviser the position at which SPO11 cuts aural the nucleosome-free region, which ability be hardly displaced on boilerplate by 30 base-pairs (half the admeasurement of 60 bp shift) from PRDM9 itself (Fig. 8b). It has been appropriate that PRDM9 generally charcoal apprenticed on the uncut chromosome14, admitting SPO11 has been proposed to be associated with DNA ends19 until or alike consecutive to fiber invasion14,19. If PRDM9 analogously charcoal apprenticed to DNA post-cleavage amid the SPO11 cut and MRE11-endonucleolytic nicking position (Fig. 8b), it could baffle with MRE11 absolution of SPO11. This would anticipate MRE11 from breeding a absolutely ssDNA overhang abandoned on one ancillary of the DSB (Fig. 8b, top). Because the accustomed on/off bounden affection of PRDM9 would actuate the affluence at which MRE11 action is blocked and SPO11-bound DNA is captured by END-seq, we would apprehend that the axial aiguille to be detected at all hotspots genome-wide, as empiric (Fig. 2b). Moreover, all hotspots had according ratios of axial aiguille to resection arresting (~ 11%), advertence that no hotspot had alternative over others, behindhand of breach frequency.

The axial arresting not abandoned reflected agee MRE11-mediated processing, but additionally appropriate DMC1- and HOP2-mediated fiber aggression and assurance with the akin chromosome arrangement (as apparent above). Attributable to this dependency, we infer that SPO11 charcoal apprenticed column homolog assurance and during the accession of a recombination boilerplate (RI). We accordingly accredit to this RI, with SPO11 capping the 3′ resected end, as SPO11-RI (Fig. 8b). Connected with our results, an absolute genomic sequencing adjustment (S1-seq) corroborates the attendance of SPO11-RI with a antipodal axial arresting polarity in WT mouse spermatocytes38.

The axial arresting in ATM-null spermatocytes is abundantly comprised of unresected SPO11cc, which accumulates MRE11 in vivo and is acute to TDP2-mediated processing (Figs. 5d, 6c, and Supplementary Fig. 4f–i). This is audible from the accessory of SPO11-RI empiric in WT cells. Elevated levels of SPO11cc in Atm–/– beef could appear from decreased MRE11 endonucleolytic or 3′−5′ exonuclease action (Fig. 5c, d), which is additionally appropriate of aggrandize Tel1 deficiency6. If Atm–/– SPO11cc reflects absolutely unresected DSBs and not SPO11-RI, then the aggregated END-seq arresting should accept no accessible polarity. To appraise this, we fiber afar the axial arresting in Atm–/– cells. Unexpectedly, the separated strands apparent a ~50 base-pair gap aural the nucleosome-depleted arena with the actual DSB polarity (Fig. 8c). If SPO11 cut already on anniversary chromatid throughout the NDR aural the citizenry of beef and remained apprenticed to DNA, there would be a 2-nt gap amid top and basal fiber DSB endpoints. We accordingly infer that the beyond gap admeasurement reflects common SPO11 double-cutting aural the aforementioned hotspot (Fig. 8c, d)44. These abstracts are connected with the added 40–70 nucleotide SPO11-oligo breed that were detected in ATM-null mice7, as two audible SPO11 cuts adjoining to one addition could absolution these best oligos alike afterwards MRE11 endonuclease action (Fig. 8d). Moreover, MRE11 ChIP-seq appear a notable dip in MRE11-binding absolutely aural the NDR (Fig. 5d, e and Supplementary Fig. 4b), connected with the accident of DNA aural hotspot centers. These after-effects are additionally accurate by abiogenetic affirmation of double-cutting in Atm–/– spermatocytes (A. Lukaszewicz, S. Keeney and M. Jasin, claimed communication).

Increased double-cutting about PRDM9-binding sites would avert it from blocking any MRE11 short-range resection that does occur, thereby abbreviation the affluence of SPO11-RIs in ATM-null beef (Fig. 8d). Our award that axial arresting in Atm–/– spermatocytes exhibits the actual polarity (Fig. 8b), and that this arresting is identical on the autosomes and non-PAR X chromosome (Supplementary Fig. 4h, i), is connected with a cogent abridgement in SPO11-RI in Atm–/– spermatocytes. Therefore, through its adjustment of SPO11 acid and resection, ATM alongside regulates the accession of SPO11-RI.

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