Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal

BRCA1 and CtIP suppress long tract gene conversion between sister chromatids

BRCA1 controls early steps of the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination, but has no known role following Rad51-mediated synapsis. Here we show that BRCA1 influences post-synaptic homologous recombination events, controlling the balance between short- (STGC) and long-tract gene conversion (LTGC) between sister chromatids. Brca1 mutant cells reveal a bias towards LTGC that is corrected by expression of wild type but not cancer-predisposing BRCA1 alleles. The LTGC bias is enhanced by depletion of CtIP but reversed by inhibition of 53BP1, implicating DNA end resection as a contributor to the STGC/LTGC balance. The impact of BRCA1/CtIP loss on the STGC/LTGC balance is abolished when the second (non-invading) end of the break is unable to support termination of STGC by homologous pairing (“annealing”). This suggests that BRCA1/CtIP-mediated processing of the second end of the break controls the annealing step that normally terminates SDSA, thereby suppressing the error-prone LTGC outcome

LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair

Leukemia/lymphoma-related factor (LRF) is a POZ/BTB and Krüppel (POK) transcriptional repressor characterized by context-dependent key roles in cell fate decision and tumorigenesis. Here we demonstrate an unexpected transcription-independent function for LRF in the classical non-homologous end joining (cNHEJ) pathway of double-strand break (DSB) repair. We find that LRF loss in cell lines and mouse tissues results in defective cNHEJ, genomic instability and hypersensitivity to ionizing radiation. Mechanistically, we show that LRF binds and stabilizes DNA-PKcs on DSBs, in turn favouring DNA-PK activity. Importantly, LRF loss restores ionizing radiation sensitivity to p53 null cells, making LRF an attractive biomarker to direct p53-null LRF-deficient tumours towards therapeutic treatments based on genotoxic agents or PARP inhibitors following a synthetic lethal strategy

Differential Regulation of Short- and Long-Tract Gene Conversion between Sister Chromatids by Rad51C

The Rad51 paralog Rad51C has been implicated in the control of homologous recombination. To study the role of Rad51C in vivo in mammalian cells, we analyzed short-tract and long-tract gene conversion between sister chromatids in hamster Rad51C(−/−) CL-V4B cells in response to a site-specific chromosomal double-strand break. Gene conversion was inefficient in these cells and was specifically restored by expression of wild-type Rad51C. Surprisingly, gene conversions in CL-V4B cells were biased in favor of long-tract gene conversion, in comparison to controls expressing wild-type Rad51C. These long-tract events were not associated with crossing over between sister chromatids. Analysis of gene conversion tract lengths in CL-V4B cells lacking Rad51C revealed a bimodal frequency distribution, with almost all gene conversions being either less than 1 kb or greater than 3.2 kb in length. These results indicate that Rad51C plays a pivotal role in determining the “choice” between short- and long-tract gene conversion and in suppressing gene amplifications associated with sister chromatid recombination

RAP80-directed tuning of BRCA1 homologous recombination function at ionizing radiation-induced nuclear foci

In response to DNA double-strand breaks (DSBs), BRCA1 forms biochemically distinct complexes with certain other DNA damage response proteins. These structures, some of which are required for homologous recombination (HR)-type DSB repair, concentrate at distinct nuclear foci that demarcate sites of genome breakage. Polyubiquitin binding by one of these structures, the RAP80/BRCA1 complex, is required for efficient BRCA1 focal recruitment, but the relationship of this process to the execution of HR has been unclear. We found that this complex actively suppresses otherwise exaggerated, BRCA1-driven HR. By controlling the kinetics by which other BRCA1-interacting proteins that promote HR concentrate together with BRCA1 in nuclear foci, RAP80/BRCA1 complexes suppress excessive DSB end processing, HR-type DSB repair, and overt chromosomal instability. Since chromosomal instability emerges when BRCA1 HR function is either unbridled or absent, active tuning of BRCA1 activity, executed in nuclear foci, is important to genome integrity maintenance

53BP1 Protects against CtIP-Dependent Capture of Ectopic Chromosomal Sequences at the Junction of Distant Double-Strand Breaks

DNA double-strand breaks (DSB) are very harmful lesions that can generate genome rearrangements. In this study, we used intrachromosomal reporters to compare both the efficiency and accuracy of end-joining occurring with close (34 bp apart) vs. distant DSBs (3200 bp apart) in human fibroblasts. We showed that a few kb between two intrachromosomal I-SceI-induced DSBs are sufficient to foster deletions and capture/insertions at the junction scar. Captured sequences are mostly coupled to deletions and can be partial duplications of the reporter (i.e., sequences adjacent to the DSB) or insertions of ectopic chromosomal sequences (ECS). Interestingly, silencing 53BP1 stimulates capture/insertions with distant but not with close double-strand ends (DSEs), although deletions were stimulated in both case. This shows that 53BP1 protects both close and distant DSEs from degradation and that the association of unprotection with distance between DSEs favors ECS capture. Reciprocally, silencing CtIP lessens ECS capture both in control and 53BP1-depleted cells. We propose that close ends are immediately/rapidly tethered and ligated, whereas distant ends first require synapsis of the distant DSEs prior to ligation. This "spatio-temporal" gap gives time and space for CtIP to initiate DNA resection, suggesting an involvement of single-stranded DNA tails for ECS capture. We therefore speculate that the resulting single-stranded DNA copies ECS through microhomology-mediated template switching

Long insertions are favored at the repair junction of unprotected distant DSEs.

<p><b>A</b>. Impact of 53BP1 and/or CtIP depletion on the size of insertions at the repair sites of distant ends (GC92 cells) or close ends (GCK20 cells) in cells transfected with control siRNA and/ or 53BP1 and/or CtIP siRNAs. For each sample, each dot represents one insertion and the red line represents the median (*: p<0.03, Mann-Whitney test). The green line indicates the threshold of 45 bp that was chosen for sequence BLAST. <b>B.</b> Impact of 53BP1 and CtIP on the frequency of long insertions (≥45 bp). Histograms represent insertions coupled to a deletion event and insertions not coupled to a deletion event. Values represent the mean +/- SEM of at least 3 independent experiments and sequencing of 78 to 190 junction sequences. (*: p = 0.02; **: p<0.005, Mann-Whitney test).</p