53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination

Callén, Elsa; Zong, Dali; Wu, Wei; Wong, Nancy; Stanlie, Andre; Ishikawa, Masatoshi; Pavani, Raphael Souza; Dumitrache, Lavinia C.; Byrum, Andrea K.; Mendez‐Dorantes, Carlos; Martínez, Paula; Canela, Andrés; Maman, Yaakov; Day, Amanda; Kruhlak, Michael J.; Blasco, Marı́a A.; Stark, Jeremy M.; Mosammaparast, Nima; McKinnon, Peter J.; Nussenzweig, André

doi:10.1016/j.molcel.2019.09.024

Cited by 95 publications

(109 citation statements)

References 57 publications

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“…In striking contrast to 53BP1-deficient somatic cells in which DSB resection is consistently and acutely increased 18,30,33 , we found that resection lengths were comparable at all meiotic hotspots in 53bp1 -/and WT spermatocytes ( Fig. 4e).…”

Section: Resultsmentioning

confidence: 54%

ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

Paiano

Yamada

et al. 2020

Nat Commun

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Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatinbound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm -/spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.

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Section: Resultsmentioning

confidence: 54%

ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

Paiano

Yamada

et al. 2020

Nat Commun

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“…We therefore performed END-seq on 53bp1 -/and BRCA1-deficient spermatocytes and measured overall resection lengths. In striking contrast to 53BP1-deficient somatic cells in which DSB resection is consistently and acutely increased 18,31,34 , we found that resection lengths were comparable at all meiotic hotspots in 53bp1 -/and WT spermatocytes ( Fig. 2d).…”

Section: Minimal Roles Of Exo1 Brca1 and 53bp1 In Long-range Resectionmentioning

confidence: 56%

“…Brca1 +/△11 mice were obtained from the NCI mouse repository. 53bp1 S25A mice were generated as described 34 . p53 -/mice were purchased from Taconic Biosciences.…”

Section: Methodsmentioning

confidence: 99%

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

Paiano

Yamada

et al. 2019

Preprint

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Meiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11. The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations ("hotspots") are determined by the DNA sequence specificity of PRDM9. Here, we demonstrate the first direct detection of meiotic DSBs and resection in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum lengths of resected DNA, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate ("SPO11-RI") present at all hotspots. The SPO11-bound intermediate is dependent on PRDM9 and caps the 3' resected end during engagement with the homologous template. We propose that SPO11-RI is generated because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm -/spermatocytes, SPO11-RI is reduced while unresected DNA-bound SPO11 accumulate because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing. MainRecombination between homologous chromosomes during meiosis requires DNA double-strand break (DSB) formation by the topoisomerase-like protein SPO11 1 . After cutting, SPO11 remains covalently bound to a two-nucleotide, 5' overhang at both ends of the DNA via phosphotyrosyl linkage. Recombination then begins with the processing of SPO11-bound DSBs into resected 3' single-stranded DNA (ssDNA) tails that preferentially invade the homologous chromosome by the recombinases DMC1 and RAD51. Studies in budding yeast Saccharomyces cerevisiae determined that the MRE11/RAD50/NBS1 (MRN) complex detects SPO11 and cooperates with Sae2 to produce a nick on the SPO11-bound strand via MRE11 endonuclease activity 2 . The nick serves as an entry point for both short-range MRE11 3'-5' exonuclease activity to degrade back to the DSB, thereby removing covalently bound SPO11 attached to a ssDNA oligonucleotide, as well as for more extensive long-range processing of 5' strands (Extended Data Fig. 1a) 2 . In budding yeast, Exo1 nuclease is uniquely responsible for this long-range 5'-3' resection 3 .Moreover, short-and long-range resection are tightly coupled in a single processive reaction (Extended Data Fig. 1a). As a result, meiotic DSBs are maximally resected as soon as they appear and unresected SPO11-bound DSBs are extremely rare 4-6 . While ATM has been shown to regulate DSB numbers and locations 7,8 , its remains unclear whether it also functions downstream in regulating SPO11 processing and resection.Distinct from yeast, DSB hotspots in mice and humans are determined by the DNA binding specificity of the PRDM9 methyltransferase 9 . Besides positioning DSBs, PRDM9...

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“…Rif1 interacts with 53BP1 and the Shieldin complex to process double strand DNA breaks, which affects the choice between homologous recombination and NHEJ. This choice plays a major role in immunoglobulin class switch recombination, and thus loss of Rif1 has dramatic consequences for immunoglobulin rearrangements (Silverman et al, 2004;Chapman et al, 2013;Callen et al, 2020) Notably, while many of these Rif1 functions in homologous recombination and NHEJ are conserved across phyla (Mattarocci et al, 2017;Fontana et al, 2018) and (reviewed in (Mattarocci et al, 2016)), the role of Rif1 in telomere length regulation is not conserved in mammals (Silverman et al, 2004;Xu and Blackburn, 2004;Buonomo et al, 2009). Rif1 regulates telomere length in a subset of species including S. pombe (Kanoh and Ishikawa, 2001) where it can interact with the telomere binding protein Taz1, and in C. glabrata (Castano et al, 2005) where it interacts with Rap1.…”

Section: Rif1 Regulation Of Origin Firing Is Conserved While Its Rolementioning

confidence: 99%

The Role of Rif1 in telomere length regulation is separable from its role in origin firing

Shubin

Greider

2020

Preprint

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To examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ∆N-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that deregulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-∆1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

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53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination

Cited by 95 publications

References 57 publications

ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

The Role of Rif1 in telomere length regulation is separable from its role in origin firing

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