ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis

The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5. These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis. Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.

The synaptonemal complex (SC) is a proteinaceous ultrastructure that forms between homologous chromosomes (homologs) during midprophase I of meiosis and plays a critical role in coordinating the repair of programmed DNA double-strand breaks (DSBs) to form cross-over (CO) products (1, 2). At the onset of leptotene, the sister chromatids are organized into linear looped chromatin arrays conjoined at the loop bases by a protein axis that runs along the chromosomes (3, 4). Early steps in the recombination pathway enable the loose alignment of homolog axes at a distance of ∼400 nm (5). Formation of the SC then initiates and continues throughout zygotene via progressive installation of transverse filaments (TFs) that run perpendicular to the aligned homolog axes (referred to as lateral elements in the context of the SC), ultimately bringing them into close apposition along their entire length at a distance of ∼100 nm (2, 5). Installation of the TFs starts at multiple synapsis initiation sites that correspond to future Class I COs in Saccharomyces cerevisiae (6). In species with larger chromosomes such as Sordaria macrospora, synapsis initiates from CO-designated sites as well as additional sites whose distribution also appears sensitive to interference (1, 5). In Arabidopsis thaliana, 20 to 25 synapsis initiation sites per cell indicate a ∼2- to 2.5-fold excess over COs and in barley 76 synapsis initiation sites, versus 17 chiasmata reveal a ∼4.5-fold excess (7, 8). Full synapsis denotes the onset of pachytene and is maintained throughout this stage during which time CO formation is completed. As prophase I progresses to diplotene/diakinesis, the SC is disassembled.TFs have been described in a variety of organisms, and in most cases, they are composed of a single protein. These include Zip1 in budding yeast, C(3)G in Drosophila melanogaster, SYCP1 in mouse, ZYP1 in A. thaliana (encoded by duplicated genes, ZYP1a and ZYP1b), ZEP1 in rice (Oryza sativa), and ZYP1 in barley (Hordeum vulgare) (9–16). Caenorhabditis elegans is an exception that possesses six TF proteins (SYP1-6) required for normal synapsis (17–22). Despite a striking lack of homology between the TFs at the primary amino acid sequence level, they share very similar structures, comprising a globular N-terminal domain linked to another globular domain at the C terminus via a long alpha helical central region that is able to form large stretches of parallel, in-register, homodimeric coiled coils (23). Studies have shown that the TFs are oriented such that the C termini are associated with lateral elements potentially interacting with DNA, while the N-terminal domains localize to the central region (2, 24). Evidence suggests that the overall three-dimensional macromolecular organization of the SC is also somewhat conserved. Analyses in mouse, Drosophila, and H. vulgare (barley) strongly suggest that these organisms form SCs with a bilayer of TFs (25–28). A multilayered structure is also supported by studies in Blabs cribrosa (beetle) (29, 30). However, key aspects of the organization of the TFs within the SC remain a matter of debate. Initially, analysis of zip1 mutants in S. cerevisiae suggested that the TFs comprise a tetramer of two opposing Zip1 dimers with their N termini forming overlapping interactions in the central region of the SC (31). X-ray crystallographic studies of the human TF, SYCP1, report that the protein forms a tetrameric building block that self-assembles into a zipper-like lattice through “head-to-head” N-terminal interactions in the SC central region and “back-to-back” interactions between adjacent C-terminal dimers at the lateral elements (24). In contrast, analysis of the mouse SC using electron tomography has led to the proposal that the SC has a more dynamic structure with TF dimers forming a variety of less regimented interactions as part of an irregular single plane. However, this model appears inconsistent with other studies in mouse which support a more ordered structure (25, 26).Mutant analysis has demonstrated that TF proteins are essential for assembly of the SC central region and thus homolog synapsis. These also confirm an important role in the control of CO formation but with some variation between organisms. Studies of zip1 mutants in S. cerevisiae have shown that the Zip1 protein is a member of the ZMM group of proteins comprising Zip1, Zip2, Zip3, Zip4, Msh4, Msh5, and Mer3 that are required for the formation of Class I interfering COs (32). CO interference is a patterning mechanism that ensures even spacing of COs along the chromosomes (33–35). In S. cerevisiae and Arabidopsis, Class I COs account for ∼85% of total COs and the remaining Class II COs (∼15%) are randomly distributed (36–38). However, in plants, Zip1 orthologs appear to be functionally independent of the other ZMM proteins for CO formation (14–16). Genetic analysis of S. cerevisiae zip1 deletion mutants revealed a modest reduction in CO formation ∼30 to 40% with residual COs no longer exhibiting CO interference leading to the suggestion that the SC may mediate this process (10). Subsequent studies based on a molecular analysis of recombination intermediates in zip1 and other zmm mutants argue against a role for the SC in mediating interference as they indicate that the fate of DSBs is designated at an early stage in the recombination pathway prior to installation of the SC (32, 39). In female Drosophila lacking the TF protein C(3)G, DSB formation is thought to be reduced and they fail to form COs, although SC formation is independent of recombination (12). These authors also report that analysis of flies expressing a mutant version of the protein reveals that a complete SC is not required for CO interference (12). A major reduction in COs of ∼90% is also observed in mouse sycp1 mutants although DSB formation appears normal (13). Similarly in C. elegans (in which SC installation occurs at pairing centers), syp-1 and syp-2 null mutants recombination is initiated but COs do not form (17, 18). A further study in which the SC central region was partially depleted by RNA interference (RNAi)–induced SYP-1 knockdown found that CO interference was reduced leading to an increase in COs, suggesting a role for the SC in limiting COs (40).TFs have been studied in several plant species including Arabidopsis, barley, and rice (14–16). Analysis of Tos17 insertion mutants of the rice TF gene ZEP1 demonstrated that in common with other organisms, it is essential for SC formation and affects CO formation (16). However, rather than displaying a reduction in COs, analysis of the short arm of chromosome 11 revealed a more than threefold increase in COs in zep1 mutants (16). Like rice, barley is a member of the grass family (Poaceae), and in common with rice, RNAi knockdown lines of the TF protein HvZYP1 are defective in SC formation, but in contrast, CO formation is reduced to ∼25% of wild-type levels (15). In Arabidopsis, the TF protein, ZYP1, is encoded by functionally redundant duplicated genes, ZYP1a and ZYP1b, which share 93% homology and are encoded within 2 kb of each other on opposite strands of chromosome 1. Individual zyp1a and zyp1b mutants are fertile and possess only mild meiotic phenotypes, and as isolation of a double mutant has thus far proved intractable, functional analysis of ZYP1 has relied on RNAi knockdown lines (14). As expected, these lines failed to assemble an SC. Chiasma frequency was reduced by ∼20 to 30% and based on metaphase I bivalent shapes, they appeared to exhibit interference, but a proportion involved ectopic recombination with nonhomologs (14).Although existing studies imply that there may be some variation in the role of the SC in relation to CO control in plants, the studies in Arabidopsis and barley were based on RNAi knockdown lines rather than TF mutants. Hence to address this issue, we have generated CRISPR/Cas zyp1a/zyp1b mutants. This has enabled a detailed analysis of ZYP1 function in Arabidopsis, revealing that it is required for formation of the obligate CO and implementation of CO patterning. Loss of the protein also disrupts the normal program of homolog coalignment during prophase I.     

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage,while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Schlafen-11 (SLFN11) is an emergent restriction factor against genomic instability acting by eliminating cells with replicative damage (1–6) and potentially acting as a tumor suppressor (6, 7). SLFN11-expressing cancer cells are consistently hypersensitive to a broad range of chemotherapeutic drugs targeting DNA replication, including topoisomerase inhibitors, alkylating agents, DNA synthesis, and poly(ADP-ribose) polymerase (PARP) inhibitors compared to SLFN11-deficient cancer cells, which are chemoresistant (1, 2, 4, 8–17). Profiling SLFN11 expression is being explored for patients to predict survival and guide therapeutic choice (8, 13, 18–24).The Cancer Genome Atlas (TCGA) and cancer cell databases demonstrate that SLFN11 mRNA expression is suppressed in a broad fraction of common cancer tissues and in ∼50% of all established cancer cell lines across multiple histologies (1, 2, 5, 8, 13, 25, 26). Silencing of the SLFN11 gene, like known tumor suppressor genes, is under epigenetic mechanisms through hypermethylation of its promoter region and activation of histone deacetylases (HDACs) (21, 23, 25, 26). A recent study in small-cell lung cancer patient-derived xenograft models also showed that SLFN11 gene silencing is caused by local chromatin condensation related to deposition of H3K27me3 in the gene body of SLFN11 by EZH2, a histone methyltransferase (11). Targeting epigenetic regulators is therefore an attractive combination strategy to overcome chemoresistance of SLFN11-deficient cancers (10, 25, 26). An alternative approach is to attack SLFN11-negative cancer cells by targeting the essential pathways that cells use to overcome replicative damage and replication stress. Along these lines, a prior study showed that inhibition of ATR (Ataxia Telangiectasia- and Rad3-related) kinase reverses the resistance of SLFN11-deficient cancer cells to PARP inhibitors (4). However, targeting the ATR pathway in SLFN11-deficient cells has not yet been fully explored.SLFN11 consists of two functional domains: A conserved nuclease motif in its N terminus and an ATPase motif (putative helicase) in its C terminus (2, 6). The N terminus nuclease has been implicated in the selective degradation of type II tRNAs (including those coding for ATR) and its nuclease structure can be derived from crystallographic analysis of SLFN13 whose N terminus domain is conserved with SLFN11 (27, 28). The C terminus is only present in the group III Schlafen family (24, 29). Its potential ATPase activity and relationship to chemosensitivity to DNA-damaging agents (3–5) imply that the ATPase/helicase of SLFN11 is involved specifically in DNA damage response (DDR) to replication stress. Indeed, inactivation of the Walker B motif of SLFN11 by the mutation E669Q suppresses SLFN11-mediated replication block (5, 30). In addition, SLFN11 contains a binding site for the single-stranded DNA binding protein RPA1 (replication protein A1) at its C terminus (3, 31) and is recruited to replication damage sites by RPA (3, 5). The putative ATPase activity of SLFN11 is not required for this recruitment (5) but is required for blocking the replication helicase complex (CMG-CDC45) and inducing chromatin accessibility at replication origins and promoter sites (5, 30). Based on these studies, our current model is that SLFN11 is recruited to “stressed” replication forks by RPA filaments formed on single-stranded DNA (ssDNA), and that the ATPase/helicase activity of SLFN11 is required for blocking replication progression and remodeling chromatin (5, 30). However, underlying mechanisms of how SLFN11 irreversibly blocks replication in DNA damage are still unclear.Increased RPA-coated ssDNA caused by DNA damage and replication fork stalling also triggers ATR kinase activation, promoting subsequent phosphorylation of CHK1, which transiently halts cell cycle progression and enables DNA repair (32). ATR inhibitors are currently in clinical development in combination with DNA replication damaging drugs (33, 34), such as topoisomerase I (TOP1) inhibitors, which are highly synergistic with ATR inhibitors in preclinical models (35). ATR inhibitors not only inhibit DNA repair, but also lead to unscheduled replication origin firing (36), which kills cancer cells (37, 38) by inducing genomic alterations due to faulty replication and mitotic catastrophe (33).The replication licensing factor CDT1 orchestrates the initiation of replication by assembling prereplication complexes (pre-RC) in G1-phase before cells enter S-phase (39). Once replication is started by loading and activation of the MCM helicase, CDT1 is degraded by the ubiquitin proteasomal pathway to prevent additional replication initiation and ensure precise genome duplication and the firing of each origin only once per cell cycle (39, 40). At the end of G2 and during mitosis, CDT1 levels rise again to control kinetochore-microtubule attachment for accurate chromosome segregation (41). Deregulated overexpression of CDT1 results in rereplication, genome instability, and tumorigenesis (42). The cellular CDT1 levels are tightly regulated by the damage-specific DNA binding protein 1 (DDB1)–CUL4^CDT2 E3 ubiquitin ligase complex in G1-phase (43) and in response to DNA damage (44, 45). How CDT1 is recognized by CUL4^CDT2 in response to DNA damage remains incompletely known.In the present study, starting with a human genome-wide RNAi screen, bioinformatics analyses, and mechanistic validations, we explored synthetic lethal interactions that overcome the chemoresistance of SLFN11-deficient cells to the TOP1 inhibitor camptothecin (CPT). The strongest synergistic interaction was between depletion of the ATR/CHK1-mediated DNA damage response pathways and DNA-damaging agents in SLFN11-deficient cells. We validated and expanded our molecular understanding of combinatorial strategies in SLFN11-deficient cells with the ATR (M4344 and M6620) and CHK1 (SRA737) inhibitors in clinical development (33, 46, 47) and found that ATR inhibition leads to CDT1 stabilization and hyperphosphorylation with mitotic catastrophe. Our study also establishes that SLFN11 promotes the degradation of CDT1 by binding to DDB1, an adaptor molecule of the CUL4^CDT2 E3 ubiquitin ligase complex, leading to an irreversible replication block in response to replicative DNA damage.     

Discriminating symbiosis and immunity signals by receptor competition in rice

Plants encounter various microbes in nature and must respond appropriately to symbiotic or pathogenic ones. In rice, the receptor-like kinase OsCERK1 is involved in recognizing both symbiotic and immune signals. However, how these opposing signals are discerned via OsCERK1 remains unknown. Here, we found that receptor competition enables the discrimination of symbiosis and immunity signals in rice. On the one hand, the symbiotic receptor OsMYR1 and its short-length chitooligosaccharide ligand inhibit complex formation between OsCERK1 and OsCEBiP and suppress OsCERK1 phosphorylating the downstream substrate OsGEF1, which reduces the sensitivity of rice to microbe-associated molecular patterns. Indeed, OsMYR1 overexpression lines are more susceptible to the fungal pathogen Magnaporthe oryzae, whereas Osmyr1 mutants show higher resistance. On the other hand, OsCEBiP can bind OsCERK1 and thus block OsMYR1–OsCERK1 heteromer formation. Consistently, the Oscebip mutant displayed a higher rate of mycorrhizal colonization at early stages of infection. Our results indicate that OsMYR1 and OsCEBiP receptors compete for OsCERK1 to determine the outcome of symbiosis and immunity signals.

In nature, plants live and interact with diverse microbes, including symbionts and pathogens. To discern friends from foes, plants have evolved various receptors that sense external microbes. Plant immune responses are triggered when pattern recognition receptors (PRRs) at the plasma membrane recognize microbe-associated molecular patterns (MAMPs) (1). MAMPs are highly conserved molecular signatures within a class of microbes and include fungal chitin, bacterial flagellin, and elongation factor Tu (EF-Tu) (2). PRRs comprise receptor-like kinases and receptor-like proteins (3). In plant–symbiont interactions, receptor kinases at the plasma membrane recognize signals that trigger symbiosis (4, 5).Arbuscular mycorrhizal (AM) fungi secrete short-chain chitooligosaccharides (COs) and nonsulfated lipochitooligosaccharides (LCOs), called mycorrhizal factors (Myc factors), that are recognized by plant receptors and mediate the establishment of AM symbiosis (6–14). In rice, perception of the AM symbiotic signal is mediated by a lysin motif (LysM)–containing receptor kinase (LYKs), OsMYR1, that directly binds to CO4 and subsequently interacts with OsCERK1 (8). Interestingly, OsCERK1 is also a well-known receptor involved in MAMP-triggered immunity (15–17). In rice, an OsCERK1–OsCEBiP receptor complex recognizes chitin and triggers immune responses (18). Additionally, OsCERK1 interacts with OsLYP4 and OsLYP6 to participate in peptidoglycan perception (19). Thus, OsCERK1 is a node that crosses immunity and symbiosis.Fungal cell walls consist of about 1 to 20% chitin, which is a long-chain polymer of N-acetylglucosamine. To protect themselves from fungal infection, plants secrete chitinases that break down chitin and release COs (20). Long-chain COs are recognized by specific receptors and trigger immunity, whereas short-chain COs are associated with non-stress–related plant responses (21). Similarly, in mammals, shorter oligomers induce a weaker defense response than longer oligomers (22). Intriguingly, although chitin is the principal component of AM fungi, AM symbiosis triggers only a weak defense response (23, 24). Moreover, pretreatment of plants with CO4 also suppresses their defense response (21), implying that CO4 and OsMYR1 might suppress defense responses during AM symbiosis. However, the mechanism remains unclear.Interestingly, a recent study reported that COs ranging from four to eight residues in length (CO4 to CO8) can serve as symbiotic signals in Medicago truncatula, although CO8 is typically considered an immunity signal (9, 25). In rice, LCOs cannot induce symbiotic calcium oscillations, and short-chain COs are the major symbiotic signals from mycorrhizal fungi (26). In this study, we found that the shorter-chain chitooligosaccharide CO4 and its receptor OsMYR1 can suppress immune signaling induced by CO8 in rice. Our data indicate that the balanced perception of CO4 and CO8 by the symbiotic receptor OsMYR1, and the MAMP receptor OsCEBiP is crucial for the establishment of AM symbiosis in rice.     

Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.

Homologous recombination (HR) between maternal and paternal chromosomes is the central theme of meiosis because it generates genetic diversity among haploid gametes and, upon fertilization, among offspring. Meiotic recombination is initiated via programmed double-strand breaks (DSBs), which are preferentially repaired by HR using a homologous nonsister chromosome (interhomolog) rather than a sister chromatid (intersister) as template. Ultimately, the interhomolog DSB repair pathway results in two types of recombination products, cross-overs (COs) and noncross-overs (NCOs). COs involve the exchange of flanking markers and possible gene conversion (GC), whereas NCOs are GC events without exchange of flanking markers. In Saccharomyces cerevisiae, the synthesis-dependent strand annealing repair pathway is responsible for the formation of most NCO products, whereas CO products are formed by resolution of Holliday junction intermediates. Chiasma, which are the cytological manifestations of COs, provide a physical link to hold the parental chromosomes together as a bivalent until anaphase I, thereby ensuring proper segregation of parental chromosomes during the first meiotic division (MI) (1–4).A key step in HR is alignment and pairing of homologous DNAs. The RecA-like recombinases initiate pairing by binding to single-strand DNA (ssDNA) to form a helical nucleoprotein filament. This nucleoprotein filament, often referred to as the presynaptic complex, can pair with double-strand DNA (dsDNA) to yield synaptic complexes consisting of triplex-helical DNA pairing intermediates (5). Sexual eukaryotes fall into two groups with respect to their RecA-like recombinases. The first group (referred to as “dual-RecA eukaryotes”) includes budding and fission yeast, higher plants, mammals, and some basidiomycete fungi. The ubiquitous Rad51 (as the only RecA-like enzyme) is responsible for HR during mitosis or vegetative growth in both groups. Rad51 also collaborates with meiosis-specific Dmc1 in the dual-RecA eukaryotes to catalyze interhomolog recombination during meiosis (5). Interestingly, Dmc1 was lost from the second group, which includes Drosophila melanogaster, Caenorhabditis elegans, and some Pezizomycotina filamentous fungi (e.g., Neurospora crassa and Sodaria macrospora). It was reported previously that Dmc1 in budding yeast, fission yeast, and mammals is superior to Rad51 in tolerating sequence mismatches during the formation of triplex-helical DNA pairing intermediates (6–10). Consistent with this hypothesis, S. cerevisiae Dmc1 (ScDmc1)-mediated recombination is more efficient than ScRad51-mediated recombination in highly polymorphic diploid hybrid yeasts (e.g., SK1/S288c and YJM/S288c) (11). The absence of Dmc1 in the “Rad51-only” organisms raises the intriguing question as to how interhomolog recombination is possible among zygotes with highly diversified genome sequences.Trichoderma reesei, like N. crassa, is a Pezizomycotina filamentous fungus. The wild-isolate strain QM6a was originally isolated from one of the Solomon Islands during the Second World War. It is the ancestor of all T. reesei workhorse strains currently used for industrial production of lignocellulosic biomass-degrading enzymes and recombinant proteins (12). Sexual mating of T. reesei, also like that of N. crassa, requires female development, a process by which a female vegetative hypha first forms an ascogonium and then develops into a protoperithecium. The protoperithecium contains a specialized hypha called the trichogyne that is attracted to a male cell, commonly a conidium (the asexual spore), with which it undergoes cell fusion to generate a zygote (13–15). Trichogyne–conidial mating results in maternal transmission of mitochondrial genomes from the female trichogyne parent in these ascomycete fungi. Due to gene mutations in the MAP kinase scaffold protein HAM5 (IDC1) required for female development and vegetative hyphal fusion (anastomosis) (15), the wild isolate QM6a is female sterile. That is why QM6a was thought to be an asexual filamentous fungus for a long time. A milestone in this respect was the finding that QM6a has a MAT1-2 locus and that it could mate with a female fertile Hypocrea jacorina CBS999.97(MAT1-1) strain (13). CBS999.97, the teleomorph (sexual reproductive stage) of QM6a, was sampled from French Guiana (16). CBS999.97(MAT1-1) and CBS999.97(MAT1-2) were derived from two ascospores (i.e., the sexual spores) generated by a CBS999.97 fruiting body, respectively (13). Crosses of CBS999.97(MAT1-1) with QM6a or CBS999.97(MAT1-2) can induce rapid sexual development under laboratory conditions and generate reproductive structures. The round-shaped stromata (fruiting bodies) are composed of a thallus containing multiple flask-shaped perithecia. Each perithecium contains a bouquet of linear asci (13). The 16 ascospores in each ascus are generated from meiosis, followed by two rounds of postmeiotic mitosis. Accordingly, these 16 ascospores can be divided into four different groups and each group contains four genetically identical ascospores (17).T. reesei possesses only one copy of a RecA-like recombinase gene (rad51 but not dmc1) (18). In this study, we first show that the genome sequences of QM6a and CBS999.97 possess high sequence heterogeneity. T. reesei Rad51 (TrRad51) proteins are essential for DSB repair during vegetative growth, as well as for interhomolog recombination during meiosis. We also report that TrRad51, like ScDmc1 but not ScRad51, can stabilize mismatches during the formation of triplex-helical DNA pairing intermediates, thereby promoting interhomolog recombination during hybrid meiosis of QM6a and CBS999.97(MAT1-1).     

Ku70 suppresses alternative end joining in G1-arrested progenitor B cells

Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) throughout interphase but predominates in G1 phase when homologous recombination is unavailable. Complexes containing the Ku70/80 (“Ku”) and XRCC4/ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Lig4 are absolutely required for joining RAG1/2 endonuclease (“RAG”)-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.

DNA double-strand breaks (DSBs) arise from sources both intrinsic and extrinsic to the cell, and improper DSB repair can lead to genomic instability and oncogenic translocations. To resolve DSBs, mammalian cells largely use two major classes of repair pathways: classical nonhomologous end joining (C-NHEJ), which is active throughout interphase, and homology-directed repair (HDR), which is only active in S/G2 cell cycle phases (1, 2). In the absence of C-NHEJ, cycling cells have been found to also join DSBs by an alternative end-joining pathway or pathways (1).Programmed, cell-intrinsic DSBs are generated during V(D)J recombination in developing B and T lymphocytes. V(D)J recombination assembles V, D, and J gene segments into variable region exons within antigen receptor loci of lymphocyte progenitors during the G1 cell cycle phase (3). The RAG1/2 (RAG) endonuclease is recruited to a recombination center in antigen receptor loci (3, 4), where it binds recombination signal sequence (RSS) located adjacent to V, D, and J gene segments in one of its two active sites (5, 6). Then, the single RSS-bound RAG linearly scans long-range distances of adjacent chromatin in the locus, presented by cohesin-mediated loop extrusion, for compatible RSSs with which to mediate cleavage (4, 7–13). Once two RSSs are appropriately paired, RAG cleaves between the two sets of RSSs and their coding ends (CEs) to form RSS and CE DSB ends that are held in a postcleavage synaptic complex (14). Joining of cleaved RSS ends to each other and coding ends to each other, respectively, is subsequently carried out exclusively by C-NHEJ (15, 16), potentially due to RAG shepherding the broken ends specifically into the C-NHEJ pathway (17, 18). V(D)J recombination end-specific joining is unlike most chromosomal translocations or deletions (involving, for example, Cas9:gRNA or other types of DSBs) in which a given DSB end can join to either DSB end of the other DSB (19, 20).C-NHEJ contains the “core” factors Ku70/Ku80 (Ku), which form the DSB recognition complex, as well as XRCC4/ligase IV (Lig4), which forms the DSB ligation complex. Core C-NHEJ factors are necessary for joining CEs and RSS ends during V(D)J recombination in G1-phase developing lymphocyte progenitors; accordingly, mice deficient in core C-NHEJ factors exhibit a severe combined immune deficiency (SCID) due to defective repair during V(D)J recombination (15, 16). However, Ku70-deficient mice can have a “leaky” SCID when compared to a complete SCID in XRCC4/Lig4-deficient mice, consistent with a low level of V(D)J recombination-like joining in the absence of Ku (21). Deficiency in core C-NHEJ factors also leads to substantial p53-dependent apoptosis of newly generated postmitotic neurons (22–24); however, the impact of Ku deficiency on neuronal apoptosis is not nearly as severe as that of XRCC4 or Lig4 deficiency (25).As defined in the context of core C-NHEJ deficiency, cycling mammalian cells can also access an alternative end-joining (A-EJ) pathway (or pathways) to relatively robustly join DSB ends generated via translocations or during immunoglobulin heavy chain (IgH) class switch recombination (CSR) in mature B cells (26–28). A-EJ also joins chromosomally I-SceI-generated DSBs in cycling mammalian cell lines (29, 30), fuses dysfunctional telomeres (31), and promotes translocations to replication stress-enhanced recurrent DSB clusters in neural stem and progenitor cells (32). Implicated A-EJ factors include: Parp1, XRCC1/ligase III (31, 33), Pol θ (34, 35), and RAD52 (36). Recent studies have also implicated Pol θ as specifically involved in A-EJ in the S/G2 phase (37). Cumulatively, studies of A-EJ have not fully addressed all contexts through which cells commit to A-EJ versus C-NHEJ (37–43). In the context of V(D)J recombination, the postsynaptic RAG complex itself has been implicated in shepherding V(D)J RSS and coding end DSBs into the C-NHEJ versus A-EJ pathways in G1-phase progenitor B cell lines (16–18). However, whether RAG-generated DSBs can translocate to more general DSBs or whether repair of general DSBs in G1-arrested progenitor B cells can employ A-EJ have remained to be determined (37).XRCC4- or Lig4-deficient mice succumb to embryonic lethality, which along with their severe neuronal apoptosis is rescued by p53 deficiency, with rescue of neuronal development and embryonic development potentially occurring by rescue of newly generated Lig4- and XRCC4-deficient neurons from p53-dependent apoptosis in the presence of large numbers of unrepaired DSBs (22, 23). In contrast, Ku-deficient mice do not have an embryonic lethal phenotype and, correspondingly, exhibit a much milder neuronal apoptosis phenotype (21, 25, 44). Notably, Ku deficiency rescues the embryonic lethality of Lig4-deficiency mice and has related effects in cell lines (45, 46). In this context, Ku binding to unrepaired breaks in the context of Lig4- or XRCC4-deficient newly generated neurons has been speculated to suppress their ability to repair persistent DSBs by A-EJ, thereby promoting their apoptotic cell death (15, 47). As DSBs can be substantially joined by A-EJ in cycling Lig4- or XRCC4-deficient cells (26, 29), such Ku-dependent down-regulation of A-EJ could in theory have more impact in noncycling cells such as neurons and G1-phase progenitor B cells.To ascertain whether additional mechanisms that might restrict joining of RAG-initiated DSBs and determine whether joining of other types of DSBs is also restricted in the G1 cell cycle phase, we mapped RAG-, Cas9:gRNA- and Zinc finger nuclease-generated DSB repair fates in G1-arrested v-Abl-transformed progenitor B cell lines through a version of our linear amplification-mediated, high-throughput, genome-wide translocation sequencing (LAM-HTGTS) (48), modified to map “bait” DSB rejoining at single nucleotide resolution.     

Rtt105 promotes high-fidelity DNA replication and repair by regulating the single-stranded DNA-binding factor RPA

Single-stranded DNA (ssDNA) covered with the heterotrimeric Replication Protein A (RPA) complex is a central intermediate of DNA replication and repair. How RPA is regulated to ensure the fidelity of DNA replication and repair remains poorly understood. Yeast Rtt105 is an RPA-interacting protein required for RPA nuclear import and efficient ssDNA binding. Here, we describe an important role of Rtt105 in high-fidelity DNA replication and recombination and demonstrate that these functions of Rtt105 primarily depend on its regulation of RPA. The deletion of RTT105 causes elevated spontaneous DNA mutations with large duplications or deletions mediated by microhomologies. Rtt105 is recruited to DNA double-stranded break (DSB) ends where it promotes RPA assembly and homologous recombination repair by gene conversion or break-induced replication. In contrast, Rtt105 attenuates DSB repair by the mutagenic single-strand annealing or alternative end joining pathway. Thus, Rtt105-mediated regulation of RPA promotes high-fidelity replication and recombination while suppressing repair by deleterious pathways. Finally, we show that the human RPA-interacting protein hRIP-α, a putative functional homolog of Rtt105, also stimulates RPA assembly on ssDNA, suggesting the conservation of an Rtt105-mediated mechanism.

Faithful DNA replication and repair are essential for the maintenance of genetic material (1). Even minor defects in replication or repair can cause high loads of mutations, genome instability, cancer, and other diseases (1). Deficiency in different DNA repair or replication proteins can lead to distinct mutation patterns (2–4). For example, deficiency in mismatch repair results in increased microsatellite instability, while deficiency in homologous recombination repair is often associated with tandem duplications or deletions (3–7). Sequence analysis of various cancer types has identified many distinct genome rearrangement and mutation signatures (8). However, the genetic basis for some of these signatures remains poorly understood, thus requiring further investigation in experimental models (8).In eukaryotic cells, Replication Protein A (RPA), the major single-stranded DNA (ssDNA) binding protein complex, is essential for DNA replication, repair, and recombination (9–13). It is also crucial for the suppression of mutations and genome instability (14–17). RPA acts as a key scaffold to recruit and coordinate proteins involved in different DNA metabolic processes (14, 15, 17). As the first responder of ssDNA, RPA participates in both replication initiation and elongation (10, 12, 13). During replication or under replication stresses, the exposed ssDNA must be protected and stabilized by RPA to prevent formation of secondary structures (14, 16). RPA is also essential for DNA double-stranded break (DSB) repair by the homologous recombination (HR) pathway (18–21). During HR, the 5′-terminated strands of DSBs are initially processed by the resection machinery, generating 3′-tailed ssDNA (22). The 3′-ssDNA becomes bound by the RPA complex to activate the DNA damage checkpoint (23). RPA is subsequently replaced by the Rad51 recombinase to form a Rad51 nucleoprotein filament (19, 24). This recombinase filament catalyzes invasion of the 3′-strands at the homologous sequence to form the D-loop structure, followed by repair DNA synthesis and resolution of recombination intermediates (18, 19, 24). During HR, RPA prevents the formation of DNA secondary structures and protects 3′-ssDNA from nucleolytic degradation (25). In addition, recent work implies a role of RPA in homology recognition (26).RPA is composed of three subunits, Rfa1, Rfa2, and Rfa3, and with a total of six oligonucleotide-binding (OB) motifs that mediate interactions with ssDNA or proteins (14, 17, 27). RPA can associate with ssDNA in different modes (28). It binds short DNA (8 to 10 nt) in an unstable mode and longer ssDNA (28 to 30 nt) in a high-affinity mode (28–31). Recent single-molecule studies revealed that RPA binding on ssDNA is highly dynamic (28, 32). It can rapidly diffuse within the bound DNA ligand and quickly exchange between the free and ssDNA-bound states (32–35). The cellular functions of RPA rely on its high ssDNA-binding affinity and its ability to interact with different proteins (28). Although RPA has a high affinity for ssDNA, recent studies have suggested that the binding of RPA on chromatin requires additional regulations (36). How RPA is regulated to ensure replication and repair fidelity remains poorly understood.Rtt105, a protein initially identified as a regulator of the Ty1 retrotransposon, has recently been shown to interact with RPA and acts as an RPA chaperone (36). It facilitates the nuclear localization of RPA and stimulates the loading of RPA at replication forks in unperturbed conditions or under replication stresses (36). Rtt105 exhibits synthetic genetic interactions with genes encoding replisome proteins and is required for heterochromatin silencing and telomere maintenance (37). The deletion of RTT105 results in increased gross chromosomal rearrangements and reduced resistance to DNA-damaging agents (36, 38). In vitro, Rtt105 can directly stimulate RPA binding to ssDNA, likely by changing the binding mode of RPA (36).In this study, by using a combination of genetic, biochemical, and single-molecule approaches, we demonstrate that Rtt105-dependent regulation of RPA promotes high-fidelity genome duplication and recombination while suppressing mutations and the low-fidelity repair pathways. We provide evidence that human hRIP-α, the putative functional homolog of yeast Rtt105, could regulate human RPA assembly on ssDNA in vitro. Our study unveils a layer of regulation on the maintenance of genome integrity that relies on dynamic RPA binding on ssDNA to ensure high-fidelity replication or recombination.