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Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response to a low dose of CPT. At a high concentration of CPT, CSB is required to promote the restart of DNA replication through MUS81–RAD52–POLD3-dependent break-induced replication (BIR). In the absence of CSB, resumption of DNA synthesis at a high concentration of CPT can occur through POLQ–LIG3-, LIG4-, or PRIMPOL-dependent pathways, which are inhibited, respectively, by RAD51, BRCA1, and BRCA2 proteins. POLQ and LIG3 are core components of alternative end joining (Alt-EJ), whereas LIG4 is a core component of nonhomologous end joining (NHEJ). These results suggest that CSB regulates fork restart pathway choice following high-dosage CPT-induced fork stalling, promoting BIR but inhibiting Alt-EJ, NHEJ, and fork repriming. We find that loss of CSB and BRCA2 is a toxic combination to genomic stability and cell survival at a high concentration of CPT, which is likely due to accumulation of ssDNA gaps, underscoring an important role of CSB in regulating the therapy response in cancers lacking functional BRCA2. [ABSTRACT FROM AUTHOR]

Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response to a low dose of CPT. At a high concentration of CPT, CSB is required to promote the restart of DNA replication through MUS81–RAD52–POLD3-dependent break-induced replication (BIR). In the absence of CSB, resumption of DNA synthesis at a high concentration of CPT can occur through POLQ–LIG3-, LIG4-, or PRIMPOL-dependent pathways, which are inhibited, respectively, by RAD51, BRCA1, and BRCA2 proteins. POLQ and LIG3 are core components of alternative end joining (Alt-EJ), whereas LIG4 is a core component of nonhomologous end joining (NHEJ). These results suggest that CSB regulates fork restart pathway choice following high-dosage CPT-induced fork stalling, promoting BIR but inhibiting Alt-EJ, NHEJ, and fork repriming. We find that loss of CSB and BRCA2 is a toxic combination to genomic stability and cell survival at a high concentration of CPT, which is likely due to accumulation of ssDNA gaps, underscoring an important role of CSB in regulating the therapy response in cancers lacking functional BRCA2.

Alternative end joining (A-EJ) catalyzes substantial level of antibody class switch recombination (CSR) in B cells deficient for classical non-homologous end joining, featuring increased switch (S) region DSB resection and junctional microhomology (MH). While resection has been suggested to initiate A-EJ in model DSB repair systems using engineered endonucleases, the contribution of resection factors to A-EJ-mediated CSR remains unclear. In this study, we systematically dissected the requirement for individual DSB resection factors in A-EJ-mediated class switching with a cell-based assay system and high-throughput sequencing. We show that while CtIP and Mre11 both are mildly required for CSR in WT cells, they play more critical roles in mediating A-EJ CSR, which depend on the exonuclease activity of Mre11. While DNA2 and the helicase/HRDC domain of BLM are required for A-EJ by mediating long S region DSB resection, in contrast, Exo1’s resection-related function does not play any obvious roles for class switching in either c-NHEJ or A-EJ cells, or mediated in an AID-independent manner by joining of Cas9 breaks. Furthermore, ATM and its kinase activity functions at least in part independent of CtIP/Mre11 to mediate A-EJ switching in Lig4-deficient cells. In stark contrast to Lig4 deficiency, 53BP1-deficient cells do not depend on ATM/Mre11/CtIP for residual joining. We discuss the roles for each resection factor in A-EJ-mediated CSR and suggest that the extent of requirements for resection is context dependent.

Summary: Robust alternative end joining (A-EJ) in classical non-homologous end joining (c-NHEJ)-deficient murine cells features double-strand break (DSB) end resection and microhomology (MH) usage and promotes chromosomal translocation. The activities responsible for removing 3′ single-strand overhangs following resection and MH annealing in A-EJ remain unclear. We show that, during class switch recombination (CSR) in mature mouse B cells, the structure-specific endonuclease complex XPF-ERCC1SLX4, although not required for normal CSR, represents a nucleotide-excision-repair-independent 3′ flap removal activity for A-EJ-mediated CSR. B cells deficient in DNA ligase 4 and XPF-ERCC1 exhibit further impaired class switching, reducing joining to the resected S region DSBs without altering the MH pattern in S-S junctions. In ERCC1-deficient A-EJ cells, 3′ single-stranded DNA (ssDNA) flaps that are generated predominantly in S/G2 phase of the cell cycle are susceptible to nuclease resolution. Moreover, ERCC1 promotes c-myc-IgH translocation in Lig4−/− cells. Our study reveals an important role of the flap endonuclease XPF-ERCC1 in A-EJ and oncogenic translocation in mouse B cells.

Immunoglobulin (Ig) class switch recombination (CSR) is the process occurring in mature B cells that diversifies the effector component of antibody responses. CSR is initiated by the activity of the B cell-specific enzyme activation-induced cytidine deaminase (AID), which leads to the formation of programmed DNA double-strand breaks (DSBs) at the Ig heavy chain (Igh) locus. Mature B cells use a multilayered and complex regulatory framework to ensure that AID-induced DNA breaks are channeled into productive repair reactions leading to CSR, and to avoid aberrant repair events causing lymphomagenic chromosomal translocations. Here, we review the DNA repair pathways acting on AID-induced DSBs and their functional interplay, with a particular focus on the latest developments in their molecular composition and mechanistic regulation. Mature B cells rely on a multilayered regulatory framework to ensure that S region DSBs are preferentially channeled into the NHEJ pathway. The structure of AID-induced breaks influences both the DSB end-processing mode and end-joining pathway choice. Both the SSA factor RAD52 and the A-EJ protein HMCES contribute strand pairing activities during repair of S region DSBs. Repair of AID-initiated breaks is influenced by Igh locus-specific organizational features that ensure productive end-joining events leading to CSR. 53BP1 contributes both structural and resection modulatory roles to the regulation of CSR repair outcomes. Shieldin and CST complexes are 53BP1 and Rif1 downstream effectors, and actively counteract the processing of S region DSBs into ssDNA by combining inhibition of DNA end resection and limited fill-in synthesis of resected tracks. [ABSTRACT FROM AUTHOR]

Abstract The poly (adenosine diphosphate (ADP)-ribosyl) polymerase inhibitors (PARPi) selectively kill cancer cells with BRCA1 or BRCA2 (BRCA)-mutations. It has been proposed that cell death induction after PARPi depends on unrepaired double strand breaks (DSBs) that accumulate due to the homologous recombination deficiency of BRCA-mutated cells. Such accumulation of DSBs is inferred mainly from the high levels of DNA damage markers like phosphorylated histone H2AX. Herein, we developed a model of isogenic cell lines to show that depletion of BRCA causes PARPi-triggered cell death, replication stress (phosphorylated-H2AX and 53BP1 foci), and genomic instability. However, persistent DSBs accumulation was not detected under the same experimental conditions. Hence, at least in this cellular model, the trigger for cell death in PARPi-treated BRCA-depleted samples is not the accumulation of unrepaired DSBs. Instead, cell death better correlates with a rapid and aberrant resolution of DSBs by error-prone pathways that leads to severe chromosomic aberrations. Therefore, our results suggest that in PARPi-treated BRCA-deficient cells, chromosome aberrations may dually trigger both genomic instability and cell death.

Deficiency of either of the two homologs of poly(ADP‐ribose) glycohydrolase (PARG), PARG‐1 and PARG‐2, in Caenorhabditis elegans leads to hypersensitivity to ionizing radiation (IR). In the germ cells of parg‐2 mutant worms, the dissipation of recombinase RAD‐51 foci was slower than in wild‐type (WT) cells, suggesting defects in DNA double‐strand break (DSB) repair via homologous recombination (HR). Nevertheless, RPA‐1, the large subunit of replication protein A, accumulated faster in parg‐2 worms and disappeared earlier than in WT worms. This accelerated RPA‐1 accumulation may result from the enhanced expression of exonuclease‐1 (EXO‐1) after IR treatment. Accordingly, an exo‐1 mutation reduced IR sensitivity and accumulation of RPA‐1 in parg‐2 worms. A mutation of polq‐1, encoding for a key factor in the alternative end‐joining (Alt‐EJ) pathway, suppressed the IR hypersensitivity phenotype of parg‐2 worms and normalized the kinetics of RAD‐51 dissipation. This indicates that error‐prone Alt‐EJ may mediate DSB repair in parg‐2 worms, causing hypersensitivity to IR. In summary, PARG‐2 deficiency in C. elegans causes hyperactive DSB end resection likely through EXO‐1 overproduction. DSBs with long single‐stranded DNA ends in parg‐2 worms are thought to be repaired by Alt‐EJ instead of HR, causing genomic instability. [ABSTRACT FROM AUTHOR]

• p300 is a factor that is essential for multiple DNA damage repair pathways. • P300 inhibition makes DSB repair more mutagenic. • p300 inhibition leads to delayed DNA damage repair in pancreatic cancer cells. • P300 inhibition combined with chemotherapy leads to increased cell death. Pancreatic cancer is a notoriously deadly disease with a five-year survival rate around 10 percent. Since early detection of these tumors is difficult, pancreatic cancers are often diagnosed at advanced stages. At this point, genotoxic chemotherapeutics can be used to manage tumor growth. However, side effects of these drugs are severe, limiting the amount of treatment that can be given and resulting in sub-optimal dosing. Thus, there is an urgent need to identify chemo-sensitizing agents that can lower the effective dose of genotoxic agents and as a result reduce the side effects. Here, we use transformed and non-transformed pancreatic cell lines to evaluate DNA repair inhibitors as chemo-sensitizing agents. We used a novel next generation sequencing approach to demonstrate that pancreatic cancer cells have a reduced ability to faithfully repair DNA damage. We then determine the extent that two DNA repair inhibitors (CCS1477, a small molecule inhibitor of p300, and ART558, a small molecule inhibitor of polymerase theta) can exploit this repair deficiency to make pancreatic cancer cells more sensitive to cisplatin, a commonly used genotoxic chemotherapeutic. Immunofluorescence microscopy and cell viability assays show that CCS1477 delayed repair and significantly sensitized pancreatic cancer cells to cisplatin. The increased toxicity was not seen in a non-transformed pancreatic cell line. We also found that while ART558 sensitizes pancreatic cancer cells to cisplatin, it also sensitized non-transformed pancreatic cancer cells. [ABSTRACT FROM AUTHOR]

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD, but not DNA-PKcs-/-, B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sμ-Sγ1 junctions from DNA-PKcs-/- B cells both display similar preferences for small (2-6 nt) microhomologies (MH). In DNA-PKcs-/- cells, Sμ-Sγ1 joints are more resistant to inversions and extensive resection than Sμ-Se and Sμ-Sμ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR. [ABSTRACT FROM AUTHOR]

DNA repair deficiency is a common hallmark of many cancers and is increasingly recognised as a target for cancer therapeutics. Selecting patients for these treatments requires a functional assessment of multiple redundant DNA repair pathways. With the advent of whole-genome sequencing of cancer genomes, it is increasingly recognised that multiple signatures of mutational and chromosomal alterations can be correlated with specific DNA repair defects. The clinical relevance of this approach is underlined by the use of poly-(ADP-ribose) polymerase inhibitors (PARPi) in homologous recombination (HR) deficient high-grade serous ovarian cancers. Beyond deleterious mutations in HR-related genes such as BRCA1/2, it is recognised that HR deficiency endows ovarian cancers with specific signatures of base substitutions and structural chromosomal variation. Multiple metrics quantifying loss-of-heterozygosity (LOH) events were proposed and implemented in trials with PARPi. However, it was shown that some of the HR-deficient cases, i.e. CDK12–mutated tumours, were not associated with high LOH-based scores, but with distinct patterns of genomic alterations such as tandem duplication. Therefore, more complex signatures of structural genomic variation were identified and quantified. Ultimately, optimal prediction models for treatments targeting DNA repair will need to integrate multiples of these genomic signatures and will also need to assess multiple resistance mechanisms such as genomic reversion events that partially or fully re-activate DNA repair. [ABSTRACT FROM AUTHOR]

Activation of the Fanconi anemia (FA) pathway after treatment with mitomycin C (MMC) is essential for preventing chromosome translocations termed "radials." When replication forks stall at MMC-induced interstrand crosslinks (ICLs), the FA pathway is activated to orchestrate ICL unhooking and repair of the DNA break intermediates. However, in FA-deficient cells, how ICL-associated breaks are resolved in a manner that leads to radials is unclear. Here, we demonstrate that MMC-induced radials are dependent on DNA polymerase theta (POLθ)-mediated alternative end joining (A-EJ). Specifically, we show that radials observed in FANCD2 −/− cells are dependent on POLθ and DNA ligase III and occur independently of classical non-homologous end joining. Furthermore, treatment of FANCD2 −/− cells with POLθ inhibitors abolishes radials and leads to the accumulation of breaks co-localizing with common fragile sites. Uniformly, these observations implicate A-EJ in radial formation and provide mechanistic insights into the treatment of FA pathway-deficient cancers with POLθ inhibitors. [Display omitted] • MMC-induced chromosome radials observed in FANCD2 −/− cells are dependent on POLθ and LIG3 • Radials observed in FA-deficient cells are not dependent on C-NHEJ factors • POLθ inhibitors suppress radials and sustain breaks that map to common fragile sites Rogers et al. establish POLθ-mediated alternative end joining (A-EJ) as the critical pathway for interstrand crosslink (ICL)-mediated radial formations, a chromosome rearrangement underlying the genome instability in Fanconi anemia (FA) cells. [ABSTRACT FROM AUTHOR]

Single-strand annealing (SSA) is a DNA double-strand break (DSB) repair pathway that uses homologous repeats to bridge DSB ends. SSA involving repeats that flank a single DSB causes a deletion rearrangement between the repeats, and hence is relatively mutagenic. Nevertheless, this pathway is conserved, in that SSA events have been found in several organisms. In this review, we describe the mechanism of SSA and its regulation, including the cellular conditions that may favor SSA versus other DSB repair events. We will also evaluate the potential contribution of SSA to cancer-associated genome rearrangements, and to DSB-induced gene targeting. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including genomic instability and cell death. Indeed, misrepair of DSBs can lead to inappropriate end-joining events, which commonly underlie oncogenic transformation due to chromosomal translocations. Typically, cells employ two main mechanisms to repair DSBs: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). In addition, alternative error-prone DSB repair pathways, namely alternative end joining (alt-EJ) and single-strand annealing (SSA), have been recently shown to operate in many different conditions and to contribute to genome rearrangements and oncogenic transformation. Here, we review the mechanisms regulating DSB repair pathway choice, together with the potential interconnections between HR and the annealing-dependent error-prone DSB repair pathways. [ABSTRACT FROM AUTHOR]

Bacteria encode reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. We found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when expressed in Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase θ and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ). We also found that a group II intron RT can function similarly in DNA repair, with reciprocal active-site substitutions showing isoleucine favors MMEJ and alanine favors primer extension in both enzymes. These DNA repair functions utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have inherent ability to function in DSBR in a wide range of organisms. [Display omitted] • A Pseudomonas group II intron-like reverse transcriptase (RT) functions in DNA repair • A group II intron-encoded RT can function similarly in DNA repair • Both RTs repair double-strand breaks via microhomology-mediated end-joining • DNA repair uses conserved structural features of non-LTR-retroelement RTs DNA-repair roles for a family of evolutionarily conserved reverse transcriptases strengthen the link between genome stability and transposable element biology. [ABSTRACT FROM AUTHOR]

X-ray repair cross complementing protein 1 (XRCC1) plays an important role in base excision DNA repair (BER) as a scaffolding protein for BER enzymes. BER is one of the basic DNA repair pathways repairing greater than 20,000 endogenous lesions per cell per day. Proper functioning of XRCC1, one of the most important players in BER, was suggested to be indispensable for effective DNA repair. Despite accumulating evidence of an important role that XRCC1 plays in maintaining genomic stability, the relationship between one of its most predominant variants, R280H (rs25489), and cancer prevalence remains ambiguous. In the current study we functionally characterized the effect of the R280H variant expression on immortal non-transformed mouse mammary epithelial C127 and human breast epithelial MCF10A cells. We found that expression of R280H results in increased focus formation in mouse C127 cells and induces cellular transformation in human MCF10A cells. Cells expressing R280H showed significantly increased levels of chromosomal aberrations and accumulate double strand breaks in the G1 cell cycle phase. Our results confirm a possible link between R280H and genomic instability and suggest that individuals carrying this mutation may be at increased risk of cancer development. [ABSTRACT FROM AUTHOR]

Abstract: Classical nonhomologous end joining (C-NHEJ) is one of the two major known pathways for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Our understanding of C-NHEJ has been derived, in significant part, through studies of programmed physiologic DNA DSBs formed during V(D)J recombination in the developing immune system. Studies of immunoglobulin heavy-chain (IgH) class-switch recombination (CSR) also have revealed that there is an “alternative” end-joining process (A-EJ) that can function, relatively robustly, in the repair of DSBs in activated mature B lymphocytes. This A-EJ process has also been implicated in the formation of oncogenic translocations found in lymphoid tumors. In this review, we discuss our current understanding of C-NHEJ and A-EJ in the context of V(D)J recombination, CSR, and the formation of chromosomal translocations. [Copyright &y& Elsevier]

Robust alternative end joining (A-EJ) in classical non-homologous end joining (c-NHEJ)-deficient murine cells features double-strand break (DSB) end resection and microhomology (MH) usage and promotes chromosomal translocation. The activities responsible for removing 3′ single-strand overhangs following resection and MH annealing in A-EJ remain unclear. We show that, during class switch recombination (CSR) in mature mouse B cells, the structure-specific endonuclease complex XPF-ERCC1SLX4, although not required for normal CSR, represents a nucleotide-excision-repair-independent 3′ flap removal activity for A-EJ-mediated CSR. B cells deficient in DNA ligase 4 and XPF-ERCC1 exhibit further impaired class switching, reducing joining to the resected S region DSBs without altering the MH pattern in S-S junctions. In ERCC1-deficient A-EJ cells, 3′ single-stranded DNA (ssDNA) flaps that are generated predominantly in S/G2 phase of the cell cycle are susceptible to nuclease resolution. Moreover, ERCC1 promotes c-myc - IgH translocation in Lig4 −/− cells. Our study reveals an important role of the flap endonuclease XPF-ERCC1 in A-EJ and oncogenic translocation in mouse B cells. [Display omitted] • XPF-ERCC1 deficiency impairs alternative end joining (A-EJ)-mediated class switching • 3′ flap endonuclease, but not NER function of XPF-ERCC1, is required for A-EJ • Loss of ERCC1 impedes joining of resected 3′ flap DSB ends • ERCC1 enhances c- myc -IgH translocation in Lig4-deficient cells Bai et al. show that the 3′ flap endonuclease XPF-ERCC1, together with SLX4, is required for alternative end joining (A-EJ)-mediated class switching in mouse B cells. XPF-ERCC1 removes 3′ single-strand overhangs formed after end resection and microhomology annealing. Moreover, ERCC1 promotes c-myc-IgH translocations in A-EJ. [ABSTRACT FROM AUTHOR]

Alternative end joining (A-EJ) catalyzes substantial level of antibody class switch recombination (CSR) in B cells deficient for classical non-homologous end joining, featuring increased switch (S) region DSB resection and junctional microhomology (MH). While resection has been suggested to initiate A-EJ in model DSB repair systems using engineered endonucleases, the contribution of resection factors to A-EJ-mediated CSR remains unclear. In this study, we systematically dissected the requirement for individual DSB resection factors in A-EJ-mediated class switching with a cell-based assay system and high-throughput sequencing. We show that while CtIP and Mre11 both are mildly required for CSR in WT cells, they play more critical roles in mediating A-EJ CSR, which depend on the exonuclease activity of Mre11. While DNA2 and the helicase/HRDC domain of BLM are required for A-EJ by mediating long S region DSB resection, in contrast, Exo1's resection-related function does not play any obvious roles for class switching in either c-NHEJ or A-EJ cells, or mediated in an AID-independent manner by joining of Cas9 breaks. Furthermore, ATM and its kinase activity functions at least in part independent of CtIP/Mre11 to mediate A-EJ switching in Lig4-deficient cells. In stark contrast to Lig4 deficiency, 53BP1-deficient cells do not depend on ATM/Mre11/CtIP for residual joining. We discuss the roles for each resection factor in A-EJ-mediated CSR and suggest that the extent of requirements for resection is context dependent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sun, Bai, Xu, Xi, Gu, Zhu, Xue, Chen and Dong.)

• HDACis promote CRISPR-induced homology-involved DNA repair pathways, including HDR and alt-EJ. • HDACis increase CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. • HDACis differentially acetylate DNA repair factors. • HDACis induce cell cycle arrest at G2/M phase. • HDACis affect CRISPR-induced genome editing results through concerted action of multiple cellular machineries. Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries. [ABSTRACT FROM AUTHOR]

HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces -deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair. • HMCES is dispensable for normal hematopoiesis in mice • HMCES deficiency causes a defect in class switch recombination (CSR) of B cells • HMCES functions in the Alt-EJ pathway of the DNA DSB repair to regulate CSR • HMCES binds and protects ssDNA overhangs to promote Alt-EJ pathway HMCES (5hmC binding, embryonic stem cell-specific protein), originally found to bind 5-hydroxymethylcytosine (5hmC), was recently reported to covalently crosslink to DNA at abasic sites. Shukla et al. show that HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells. [ABSTRACT FROM AUTHOR]

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs 5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs 5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology-all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR., Competing Interests: The authors declare no competing interest.

To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost all types of DNA damage. In human cells, this task is accomplished by DNA ligases encoded by three genes, LIG1, LIG3 and LIG4. Mutations in LIG1 and LIG4 have been identified as the causative factor in two inherited immunodeficiency syndromes. Moreover, there is emerging evidence that DNA ligases may be good targets for the development of novel anti-cancer agents. In this graphical review, we provide an overview of the roles of the DNA ligases encoded by the three human LIG genes in DNA replication and repair., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The mechanistic understanding of how DNA double-strand breaks (DSB) are repaired is rapidly advancing in part due to the advent of inducible site-specific break model systems as well as the employment of next-generation sequencing (NGS) technologies to sequence repair junctions at high depth. Unfortunately, the sheer volume of data produced by these methods makes it difficult to analyze the structure of repair junctions manually or with other general-purpose software. Here, we describe methods to produce amplicon libraries of DSB repair junctions for sequencing, to map the sequencing reads, and then to use a robust, custom python script, Hi-FiBR, to analyze the sequence structure of mapped reads. The Hi-FiBR analysis processes large data sets quickly and provides information such as number and type of repair events, size of deletion, size of insertion and inserted sequence, microhomology usage, and whether mismatches are due to sequencing error or biological effect. The analysis also corrects for common alignment errors generated by sequencing read mapping tools, allowing high-throughput analysis of DSB break repair fidelity to be accurately conducted regardless of which suite of NGS analysis software is available., (© 2018 Elsevier Inc. All rights reserved.)

In this chapter, we describe a method for the recovery and analysis of alternative end-joining (alt-EJ) DNA double-strand break repair junctions following I-SceI cutting in Drosophila melanogaster embryos. Alt-EJ can be defined as a set of Ku70/80 and DNA ligase 4-independent end-joining processes that are typically mutagenic, producing deletions, insertions, and chromosomal rearrangements more frequently than higher-fidelity repair pathways such as classical nonhomologous end joining or homologous recombination. Alt-EJ has been observed to be upregulated in HR-deficient tumors and is essential for the survival and proliferation of these cells. Alt-EJ shares many initial processing steps with homologous recombination, specifically end resection; therefore, studying alt-EJ repair junctions can provide useful insight into aborted HR repair. Here, we describe the injection of plasmid constructs with specific cut sites into Drosophila embryos and the subsequent recovery of alt-EJ repair products. We also describe different analytical approaches using this system and how amplicon sequencing can be used to provide mechanistic information about alt-EJ., (© 2018 Elsevier Inc. All rights reserved.)