Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4^−/− cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ.Mammalian genomes are subjected substantial DNA damage from both endogenous processes [e.g., DNA replication, class switching recombination (CSR), etc.] and exogenous resources (e.g., ionic radiation, DNA-damaging chemicals, etc.). Evolutionarily conserved DNA repair pathways are essential to maintain both the structure integrity and the information accuracy of the genome (1). In eukaryotes, DNA double-strand breaks (DSBs), one of the most dangerous and severe types of DNA damage, are repaired mainly by two evolutionarily conserved repair pathways: homologous repair (HR) and nonhomologous end-joining (NHEJ) (2). NHEJ directly ligates two broken DSB ends, whereas HR uses a homologous template (in most of cases, the sister chromosome) for repair. Although NHEJ is simpler and faster than HR, repair by NHEJ often leads to change of sequences in the repair junctions via deletion, insertion, and mutations. Most importantly, NHEJ can also directly ligate two distant DSBs (both in cis- and trans-chromosomes), therefore leading to chromosomal deletions and translocations that are tightly linked to the genome evolution and carcinogenesis (3).The NHEJ in eukaryotes has been extensively studied in the last two decades (4–6). Mechanistically, NHEJ could be separated into several steps. First, in the DSB binding and tethering step, the DSB ends are recognized and bound by Ku70/Ku80 heterodimers that function as docking sites for other NHEJ factors. In the next end-processing step, nuclease and DNA polymerase activities are recruited to remove damaged or mismatched nucleotides and prepare the broken ends for ligation. Finally, DNA ligase IV (Lig4) complex, consisting of DNA Lig4 and its cofactor X-ray repair cross-complementing protein 4 (XRCC4), as well as the newly identified XRCC4-interacting factor (XLF), reseals the DSBs. Both V(D)J recombination and class switching recombination (CSR) have been used as important in vivo models to study the NHEJ (7). During the V(D)J recombination, DSBs generated by recombination-activating gene 1 (RAG1)/RAG2 endonuclease are joined exclusively by NHEJ. Deficiency of core NHEJ factors in mice, such as Lig4 and XRCC4, leads to complete abolishment of V(D)J recombination and lack of mature B and T cells (8, 9). When combined with a checkpoint-deficient genetic background (e.g., p53^−/−), unresolved RAG-generated DSB ends in B and T cells in core NHEJ factor-deficient mice could result in dramatic genomic instability. In particular, oncogenic chromosome translocations lead to the development of lymphoma and leukemia in those mice (10, 11). The fact that those chromosome translocations are formed by end-joining in the absence of core NHEJ factors suggested the existence of alternative end-joining pathway(s) (A-EJ). CSR, by which mammalian mature B cells change their production of Ig constant region type from one to the other, represents an excellent model to study classic NHEJ (c-NHEJ) and A-EJ (12, 13). DSBs in the IgH class switching (S) regions induced by activation-induced cytidine deaminase (AID) are repaired by NHEJ via a loop-out and deletion mechanism (14). In the absence of c-NHEJ core factors (such as Lig4, XRCC4, and Ku70/80), significant CSR activities, mediated by A-EJ, have been observed in both animals and cell lines (12, 13). There is no doubt that A-EJ contributes to all end-joining activities in the absence of c-NHEJ. However, the contribution of A-EJ in the presence of c-NHEJ is still debatable. For example, it has been suggested that A-EJ is the main end-joining activity to catalyze chromosomal translocations in murine (15) but not in human cells (16).Although A-EJ activities have been observed in many cell types and biological processes (12, 17–19), A-EJ’s exact components and mechanisms have been still not clearly revealed and sometimes are controversial (5, 20, 21). For example, whether A-EJ is a completely independent new pathway or an alternative c-NHEJ pathway in which alternative components could substitute the missing c-NHEJ factors is still debatable. Comparing with large numbers of factors and pathways involved in the early DSB repair steps, there are only three known DNA ligases (DNA Lig1, DNA Lig3, and DNA Lig4) in mammalian cells to finish the last ligation step (22). It has been proposed that those three DNA ligases function differently in various DNA metabolism processes. Although all three mammalian DNA ligases have highly homologous catalytic cores (including DBD, AdD, and OB-Fold domains), through their distinct N- and C-terminal regions, the DNA ligases may interact with different partners, which could confer functional specificity. In DSB repair, the role of Lig4 has been mostly restricted to c-NHEJ, whereas both Lig1 and Lig3 have been suggested to mediate the A-EJ in vitro and in vivo (23–28). Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate cell lines in which Lig1 or Lig3 were completely depleted, and we tried to unequivocally reveal the ligases’ roles in A-EJ.     

Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton

Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP–tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3′-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H₂O₂-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments.Exposure to ionizing irradiation (IR) has dire consequences for the cell, because it causes the formation of radicals and reactive oxygen species (ROS) that can oxidize and damage cellular components including proteins and DNA (1), whereas protection from IR-induced radical-mediated protein oxidation can lead to significant radio resistance (2). At the DNA level, IR leads to single-stranded and double-stranded DNA breaks (DSBs), with the latter being a severe threat to cellular survival (3). To cope with DSBs that may arise physiologically and/or by genotoxic environmental impacts such as IR, the cell repairs DSBs by two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), which predominate in the G1 and G2 phase of the cell cycle, respectively, and underlie cell-cycle-dependent sensitivities to IR exposure (4, 5). In the G2 phase and in the first meiotic prophase, DSB repair is mediated by HR, in yeast meiosis addressing ∼150 DSBs (6, 7) that are formed by the Topo2-related endonuclease/transesterase enzyme SPO11 (8). Absence of DSBs and the resulting compromised spore viability (9) can be partially rescued by ionizing irradiation (10). In all, yeast cells exhibit a high resistance to IR (11–13) which is also true for cells in prophase I (10, 14). In the meiosis of numerous species, programmed Spo11-induced DSBs are instrumental for homologous chromosome search and pairing and provide the substrate for HR, generating two outcomes: noncrossovers (NCO) and crossovers (CO) that allow for homolog segregation in the meiosis I division (reviewed by refs. 15 and 16). For CO to occur, homologous chromosomes need to encounter and pair lengthwise (synapse) during first meiotic prophase (see ref. 17). Homolog pairing occurs after completion of premeiotic DNA replication. Live cell studies in the synaptic meiosis of the yeast Saccharomyces cerevisiae (2n = 32) have shown that meiotic telomeres (18), chromosomes, and bivalents undergo a striking mobility throughout the entire prophase I (19–21), which contrasts with the relative immobility of pachytene bivalents in mammalian prophase I (22). It has been found that rapid and continuous telomere and chromosome movements in budding yeast meiocytes depend on actin polymerization (18–20) and an intact meiotic telomere complex (21, 23, 24). Besides a general mobility of chromosomes throughout prophase I, single bivalents are capable to rapidly move away and return to the motile chromosome mass, a behavior termed “maverick” formation (19) or rapid chromosome movements (20, 21).Exposure to ionizing radiation induces a plethora of physicochemical effects in the irradiated cells including DNA damage (1, 3). Extensive research addressing the adverse effects of IR exposure using yeast as a model system had largely been directed toward mutation induction, DSB repair, and cell cycle effects (e.g., 11–13, 25, 26). Meiotic yeast cells exposed to 50–80 krad (500–800 Gy) X or γ irradiation have been shown to exhibit a profound reduction in cell survival, particularly when exposed in the G1 cell cycle phase that lacks a sister chromatid for repair (4). Irradiated meiotic yeast cells exhibit mutations and chromosome missegregation at meiosis I, leading to reduced sporulation (5, 10, 27). While previous studies addressed late deterministic effects in irradiated yeast cells such as DNA repair, mutations, and cell survival, we were interested in the immediate consequences of IR exposure on motile meiotic chromosomes. Bivalent mobility can be expected to promote chromosomal rearrangements, if it continues after the formation of ectopic unregulated DSBs. Chromosomal translocations have, for instance, been observed after irradiation of mitotic budding yeast cells (28) and of meiotic prophase cells of mice (29). Furthermore, meiotic chromosome mobility has been proposed to be involved in regulating (adverse) chromosomal interactions (30). To study the consequences of IR exposure on meiotic chromosome mobility we followed live bivalent movements in X-irradiated and nonirradiated yeast cells expressing the GFP-tagged version of the synaptonemal complex protein ZIP1 (19) undergoing sporulation.     

UbcH7 regulates 53BP1 stability and DSB repair

DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.Prompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2–6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7, 8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9–11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12–14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12, 16, 17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12–14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 stabilizes 53BP1, forcing cells to choose NHEJ, but not HR, to repair DSBs, which poses a significant threat to cells treated with DNA damage, especially S-phase genotoxins, such as camptothecin (CPT), a topoisomerase 1 (Top1) inhibitor. The ternary CPT-Top1-DNA complex places a roadblock in the path of advancing DNA replication forks, leading to replication fork collapse and generation of one-ended DSBs. Such one-ended DSBs require HR, but not NHEJ, to repair (8). In contrast, repair of one-ended DSBs by NHEJ leads to radial chromosomes and cell death (12–14). Therefore, stabilization of 53BP1 by UbcH7 depletion increased the sensitivity of cancers cells to CPT and other DNA damaging agents. Our data suggest a novel strategy in enhancing the anticancer effect of radiotherapy or chemotherapy through stabilizing or increasing 53BP1.     

C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion

Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.Activation-induced cytidine deaminase (AID) is essential for three different genetic events: class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM), which contribute to Ig gene diversification (1–5). Although AID generates single-strand breaks (SSBs) in the Ig genes, subsequent repair steps for CSR and GC are similar to each other but are distinct from SHM in their mechanistic properties, i.e, in (i) generation of the double-strand breaks (DSBs), (ii) recombination, and (iii) the requirement for uracil-DNA-glycosylase (UNG) for the pairing of the DSB ends (6–10). Despite the similarities between GC and CSR, their repair mechanisms have distinct features: CSR recombination requires nonhomologous end joining (NHEJ), and GC depends on homologous recombination (HR). During CSR, DSB ends normally are joined by classical NHEJ (C-NHEJ), which requires specific repair proteins such as Ku80, XRCC4, or DNA ligase IV (11, 12). In the absence of C-NHEJ, a back-up end-joining pathway called “alternative end joining” (A-EJ), which is reported to be slower and also more error prone than C-NHEJ, joins the broken DSBs ends (13). On the other hand, HR, the most common form of homology-directed repair, requires long sequence homology between donor and acceptor DNA to complete the recombination step by recruiting a distinct set of repair proteins such as RAD54, RAD52, and RAD51 to the break sites (14, 15).Various studies on AID mutations in the N-terminal or C-terminal regions (4, 8, 9, 16–19) have shown that N-terminal AID mutants are compromised for CSR and are defective in SHM, indicating that the N-terminal region of AID is required for DNA cleavage (9, 16, 19). On the other hand, the C-terminal region of AID, which contains a nuclear-export signal and is responsible for AID’s shuttling activity between the nucleus and cytoplasm, is required for CSR-specific activity but not for DNA cleavage activity and SHM (8, 16). Among the series of AID C-terminal mutants examined, two mutants show characteristic features: P20, which has an insertion of 34 amino acids at residue 182 and normal nuclear-cytoplasmic shuttling activity, and JP8Bdel, which has a 16-amino acid truncation at residue 183, accumulates in the nucleus, and shows higher DNA break activity at the donor switch (S) region (16, 17). Although several reports suggest that the C-terminal region of AID is involved in protein stability (20, 21), C-terminal mutants of AID stabilized by fusing the hormone-binding domain of estrogen receptor (ER) also show similar CSR-defective phenotypes (8). Taken together, these data suggest that the DNA cleavage activity and CSR-specific activity depend on different regions of AID (8, 19). In addition, the C-terminal region of AID is essential for the interaction of AID with poly (A)⁺ RNA via a specific cofactor (22). Because CSR requires de novo protein synthesis, we proposed that after DNA cleavage the C-terminal region of AID may be involved in the regulation of the recombination step through generation of a new protein (8, 16, 22).DSBs induced by AID during CSR ultimately are joined by the efficient DNA repair pathway that requires C-NHEJ factors such as Ku70/80 (12, 23). However, in the absence of C-NHEJ, the A-EJ pathway that relies on microhomology can join the broken DNA ends, although this pathway is associated with chromosomal translocations (11, 24). Previously, we reported that JP8Bdel enhances aberrant c-myc/IgH translocations and that it fails to carry out the efficient recombination between donor and acceptor S regions in the IgH locus (8). Therefore, it is important to examine whether the AID C-terminal mutants affect the S–S joining in CSR.In the current work we examined whether the C-terminal region of AID is involved in DNA synapse formation and recombination during CSR in CH12F3-2 and spleen B cells. We also examined the effect of AID C-terminal mutations on GC in chicken DT40 cells, which depends on HR between pseudo V genes and the downstream IgVλ region. Using these CSR- and GC-monitoring systems, we demonstrate that efficient CSR and GC require the C-terminal region of AID for the formation of DSB from SSB and subsequent end synapse. Considering these findings together, we conclude that, in addition to DNA cleavage, AID has a unique function in the generation of DSBs, which is required for S–S synapse formation and joining in CSR and recombination in GC.     

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.DNA double-strand breaks (DSBs) are toxic lesions that can be repaired by two major pathways in eukaryotes: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (1). Although HR repairs DSBs in a template-dependent, high-fidelity manner, NHEJ functions to ligate DSB ends together using no or very short (1–4 bp) homology. Recently, a new pathway was identified in eukaryotes, which uses microhomologies (MHs) to repair a DSB and does not require the central proteins used in HR (Rad51, Rad52) or NHEJ (Ku70–Ku80) (2–5). In mammalian cells, this pathway of repair is known as alternative end-joining (Alt-EJ) and is often but not always associated with MHs, whereas in budding yeast, the commensurate pathway, MH-mediated end-joining (MMEJ), will typically use 5–25 bp of MH (6, 7). These pathways are associated with genomic rearrangements, and cancer genomes show evidence of MH-mediated rearrangements (8–12). In addition, eukaryotic genomes contain many dispersed repetitive elements that can lead to genome rearrangements when recombination occurs between them (13–16). Therefore, controlling DSB repair in the human genome, which features a variety of repeats, is especially important given the fact that recombination between repetitive elements has been implicated in genomic instability associated with disease (17–20).The original characterization of Alt-EJ in mammalian cells suggested it did not represent a significant DNA repair pathway and only operated in the absence of functional HR and NHEJ pathways. More recent analyses demonstrate a physiological role of Alt-EJ during DNA repair in the presence of active HR and NHEJ pathways (2, 12, 21, 22). Furthermore, examination of I-SceI–induced translocation junctions in mammalian cells revealed the frequent presence of MHs (23, 24). NHEJ-deficient and p53-null mice develop pro–B-cell lymphomas, and nonreciprocal translocations characterized by small MHs are found at their break point junctions (25–28). Similarly, in human cancers, many translocation break point junctions contain MHs, suggesting a role for Alt-EJ in cancer development (29–31) and resistance to chemotherapy and genetic disease (32–36). Hence, the presence of many short repetitive sequences in the human genome is likely to increase rearrangements mediated by MHs following the creation of a DSB.MMEJ is a distinct DSB repair pathway that operates in the presence of functional NHEJ and HR pathways (10, 37). The genetic requirements of MMEJ are being studied in the model eukaryote Saccharomyces cerevisiae and involve components traditionally considered specific to the NHEJ (Pol λ) and HR (Rad1–Rad10, Rad59, and Mre11–Rad50–Xrs2) pathways (4, 5, 10, 38). Although being clearly independent of the central NHEJ factor Ku70–Ku80 heterodimer (10, 37), the involvement of the key HR factor Rad52 in MMEJ remains uncertain. It has been reported that Rad52 is required for MMEJ repair (4, 10, 38), whereas in another assay system Rad52 suppresses MMEJ repair (37). More recently, it has been proposed that the replication protein A (RPA) regulates pathway choice between HR and MMEJ (37). In addition, several models have been proposed that identify specific pathways that may use MHs for the repair of DNA damage (39–41). Despite current advancements in our understanding of MMEJ, the precise involvement of DNA polymerases in supporting the repair of DSBs using MHs remains poorly understood. DNA polymerase λ (also called Pol4 in budding yeast) and its human homolog Pol λ are considered to be the primary candidates for the DNA polymerases working in NHEJ and MMEJ (4, 5, 42–46). Both genetic and biochemical evidence shows that Pol δ is recruited during HR to extend Rad51-dependent recombination intermediates (47–50). Recent analysis using pol32 mutants (5, 10) implicated the Pol32 subunit of Pol δ in MMEJ. Pol32 and Pol31 were also identified as subunits of the DNA polymerase zeta complex (Pol ζ) (51, 52), but previous analysis showed no effect of rev3 mutants in MMEJ (10). REV3 encodes the catalytic subunit of Pol ζ. However, an involvement of Pol δ had not been demonstrated directly before, and it is possible that Pol32 could act in conjunction with yet another DNA polymerase.Here, we report the development of a series of interchromosomal MMEJ assays in diploid S. cerevisiae to assess the mechanisms underlying the repair of DSBs using varying MHs. We focus on diploid cells, as they represent the natural state of budding yeast, which is a diplontic organism (53). The yeast mating-type switching system represents a mechanism to return haploid yeast as efficiently as possible to diploidy (54). Using a combination of genetic, molecular, and in vivo chromatin immunoprecipitation (ChIP) experiments, we provide compelling evidence for a direct involvement of Pol δ in coordinating with Pol λ in MMEJ in budding yeast.     

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR.Antibodies change during an immune response by increasing their affinity for cognate antigen and acquiring new biological properties that reside in the constant region of the heavy chain. These changes originate from modifications in the Ig genes. Somatic hypermutation (SHM) introduces single base pair mutations over the Ig variable exon (IgV), changing the antibody affinity (1, 2). Class switch recombination (CSR) exchanges the exons encoding for the constant region of the heavy chain that defines IgM for those exons defining IgG, IgA, or IgE. This process involves the stepwise generation and subsequent repair of DNA double strand breaks (DSBs) (3, 4).Activation-induced deaminase (AID) initiates both SHM and CSR by deaminating deoxycytidine to deoxyuridine at the Ig loci (2). During CSR, removal of AID-generated deoxyuridine from opposite DNA strands at two distant switch (S) regions by either the uracil DNA-glycosylase (UNG) or components of the mismatch repair pathway initiates DNA processing leading to DSBs. These DSBs at the Igh evoke a DNA damage response and are resolved by either classical nonhomologous end joining (C-NHEJ) requiring the DSBs end-binding heterodimer Ku70/80, the scaffold protein Xrcc4, and Ligase4 (4, 5) or an ill-defined alternative end-joining (A-EJ) pathway (6) for productive CSR. CSR requires the joining of two simultaneous DSBs located far apart and deletion of the intervening chromosomal segment (3). As a side effect, CSR can also produce chromosomal translocations involving the Ig loci (7). The recombination of variable diversity joining (VDJ) gene fragments is also a long-range intrachromosomal joining, but in that case, the initiating recombination-activating gene (RAG)1/2 endonuclease protects the DNA ends and promotes C-NHEJ to prevent aberrant joining (8, 9). No analogous role of AID on DNA repair during CSR has been shown so far, although AID has been suggested to stabilize inter–S-region synapsis (10). The C terminus of AID is necessary for CSR but not SHM for unknown reasons (11, 12). This requirement might reflect a role of this domain in repair, given that C-terminally truncated AID variants still produce DSBs at the S regions in B cells (13–15). However, the fact that AID can be replaced by the yeast endonuclease I-SceI for efficient CSR in engineered mice seems to argue against its need for repair (16). Thus, it is still unclear whether AID contributes to the repair steps of CSR.AID deficiency causes a hyper-IgM immunodeficiency syndrome (HIGM2) in humans. Most HIGM2 patients carry deleterious mutations in AICDA (the AID gene), which are inherited as autosomal recessive (AR) traits (17, 18). These patients lack SHM and CSR, are susceptible to infections, and develop lymphadenopathies because of germinal center hyperplasia (17, 18). Intriguingly, a small proportion of HIGM2 patients carries only one mutated AICDA allele. There is no explanation as to why these alleles are autosomal dominant (AD), but in every case, the AD allele encodes for an AID protein missing the last 8 or 12 aa (12, 15). Because this region is necessary for CSR and because AD HIGM2 patients show normal SHM, the simplest explanation would be that AD AID variants behave as dominant negatives specifically for CSR, as suggested by the families’ pedigrees (15). We hypothesized that studying this proposed dominant-negative effect could also shed light on the role of AID C terminus and show a role of AID in late steps of CSR.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Crossovers are associated with mutation and biased gene conversion at recombination hotspots

Meiosis is a potentially important source of germline mutations, as sites of meiotic recombination experience recurrent double-strand breaks (DSBs). However, evidence for a local mutagenic effect of recombination from population sequence data has been equivocal, likely because mutation is only one of several forces shaping sequence variation. By sequencing large numbers of single crossover molecules obtained from human sperm for two recombination hotspots, we find direct evidence that recombination is mutagenic: Crossovers carry more de novo mutations than nonrecombinant DNA molecules analyzed for the same donors and hotspots. The observed mutations were primarily CG to TA transitions, with a higher frequency of transitions at CpG than non-CpGs sites. This enrichment of mutations at CpG sites at hotspots could predominate in methylated regions involving frequent single-stranded DNA processing as part of DSB repair. In addition, our data set provides evidence that GC alleles are preferentially transmitted during crossing over, opposing mutation, and shows that GC-biased gene conversion (gBGC) predominates over mutation in the sequence evolution of hotspots. These findings are consistent with the idea that gBGC could be an adaptation to counteract the mutational load of recombination.Meiotic recombination, localized in recombination hotspots, not only increases genetic diversity via the formation of new haplotypes but is also an important driver of sequence evolution. The binding sites used by the human recombination machinery involving PRDM9 (PR domain containing 9) are more eroded in humans than the same sequences in chimps, given that PRDM9 in chimps uses different binding sites (1). Moreover, regions in close vicinity to these PRDM9 binding sites also showed a significant enrichment of polymorphisms in humans (2). In addition, within- and between-species sequence diversity positively correlates with regions of high recombination activity in humans (3–7) and other eukaryotes (reviewed in refs. 8–10).One process recognized as a major evolutionary force reshaping the genomic nucleotide landscape at recombination hotspots, as shown in humans (6), chimpanzees (6, 11), mice (12), yeast (13), and metazoans (14), is GC-biased gene conversion (gBGC). In gBGC, the repair of heteroduplex tracts formed during meiotic recombination leads to the non-Mendelian segregation of alleles favoring GC over AT variants. The precise molecular mechanisms leading to gBGC have yet to be unraveled, but experimental evidence has shown that in crossovers (COs) of fission yeast, GC alleles can be overtransmitted within ∼1–2 kb in length of the double-strand break (DSB) region (13), implicating mismatch repair (15).However, it is also plausible that the higher sequence variation observed at recombination hotspots is a result of a mutagenic effect of recombination: meiotic recombination is initiated by DSBs, which are associated with an increased mutation frequency. In the nonreducing division, mitosis, genetic experiments have shown that the repair of DSBs in homologous recombination involves error-prone translesion polymerases, increasing the mutation frequency at DSB sites in Drosophila (16) and yeast (17–19). In humans as well, an error-prone polymerase (DNA polymerase θ) carries out translesion synthesis in the repair of DSBs (20). In addition to mitotic DSB repair, it was recently shown that error-prone translesion synthesis polymerases (Rev1, PolZeta, and Rad30) are also involved in the repair of DSBs in meiosis in yeast (21) and could potentially contribute to a higher mutation rate in the germ cells at recombination hotspots, which are recurrent sites for DSBs.Although high mutation rates might be an important driver of the high genetic diversity and elevated divergence in regions of high recombination (refs. 5–7, 22, and 23 and reviewed by refs. 23 and 24), the mutagenic signature in population data may be obscured by a complex interplay of other factors, including selection, demographic history, and gBGC (8–10). Therefore, to detect and quantify any elevation in mutation rates arising during meiosis, we measured the frequency of de novo mutations in a large number of single COs. We provide, for the first time to our knowledge, experimental data showing that recombination associated with COs is mutagenic in humans. In our large survey of recombination products, we also find evidence that the transmission of GC alleles is favored during crossing over and that associated gBGC is acting in opposition to the introduced mutational bias.