Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

Ig class switch recombination (CSR) requires expression of activation-induced cytidine deaminase (AID) and transcription through target switch (S) regions. Here we show that knockdown of the histone chaperone facilitates chromatin transcription (FACT) completely inhibited S region cleavage and CSR in IgA-switch-inducible CH12F3-2A B cells. FACT knockdown did not reduce AID or S region transcripts but did decrease histone3 lysine4 trimethylation (H3K4me3) at both the Sμ and Sα regions. Because knockdown of FACT or H3K4 methyltransferase cofactors inhibited DNA cleavage in H3K4me3-depleted S regions, H3K4me3 may serve as a mark for recruiting CSR recombinase. These findings revealed an unexpected evolutionary conservation between CSR and meiotic recombination.A ntigen stimulation of B lymphocytes induces the expression of activation-induced cytidine deaminase (AID), which is responsible for generation of antibody memory (1, 2). Somatic hypermutation and class switch recombination (CSR) are two genetic events that engrave antibody memory into the Ig heavychain (H) locus of the B cell genome. CSR takes place between two switch (S) regions, located upstream of the individual H constant regions (C H ) and converts the isotype from IgM to another class by bringing the specific C H region close to the H variable region (V H ) exons and looping out the intervening DNA segment (3).Gene-targeting experiments in the IgH locus have shown that active transcription through the S regions is an essential requirement of CSR (4, 5). This transcription initiates from the I promoter, located upstream of each S region, and proceeds through the I exon, the intronic S region, the C H exons, and the C H introns. The mature transcripts, designated as germline transcripts (GLTs), are generated by splicing out the S region and C H intronic sequences (3). However, it is not well understood whether the transcription itself, the transcription products, or both are important for CSR. The original chromatin-opening hypothesis suggested that transcription of the S region causes its chromatin structure to be relatively open, which increases its accessibility to a putative recombinase (6, 7). In fact, the migration of the transcription machinery accumulates positive and negative supercoil in its front and rear, respectively.During this process, R-loop formation was detected in the DNA from switching B cells by the bisulfite sensitivity assay (8). The R-loop formation was considered to support the DNA deamination hypothesis proposed for the function of AID, as the single-strand DNA can serve as an efficient substrate of cytidine deamination by AID, as demonstrated in vitro (9). This hypothesis postulates that dU generated by AID deamination is recognized as a dU/dG mismatch and excised by uracil DNA glycosylase (10). The abasic sites thus formed is then cleaved by apyrimidinic/apurinic endonuclease. It has been also proposed that dU/dG mismatches are recognized and cleaved by mismatch repair proteins such as Msh2 and Msh6.On the other hand, AID was...

Highlights d 53BP1 S25A point mutation rescues lethality of BRCA1 D11 mice without restoring HR d 53BP1 S25A mutation uncouples 53BP1 end-blocking activities pre-and post-resection d RIF1/shieldin blocks BRCA1-independent loading of RAD51 onto single-strand DNA d Shieldin and PTIP associate with 53BP1 to regulate distinct end-resection pathways

Class switch recombination (CSR) is a B cell-specific genomic alteration induced by activation-induced cytidine deaminase (AID)-dependent DNA break at the immunoglobulin heavy-chain locus, followed by repair. Although chromatin-associated factors in promoting AID-induced DNA break have been widely reported, the involvement of chromatin adaptors at the repair phase of CSR remains unknown. Here, we show that the acetylated histone reader Brd4 is critical for nonhomologous end-joining (NHEJ) repair of AID- and I-SceI-induced DNA breaks. Brd4 was recruited to the DNA break regions, and its depletion from the chromatin caused CSR impairment without affecting the DNA break generation. Inhibition of Brd4 suppressed the accumulation of 53BP1 and uracil DNA glycosylase at the switch regions, perturbed the switch junctional microhomology, and reduced Igh/c-myc translocation. We conclude that Brd4 serves as a chromatin platform required for the recruitment of repair components during CSR and general DNA damage.