Break-induced replication orchestrates resection-dependent template switching.

Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres1,2. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear3-5. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments (PICh) to capture the telomeric DNA damage response proteome during BITS1,6. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.

Structural insights into BCDX2 complex function in homologous recombination.

Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.

Homologous recombination-deficient mutation cluster in tumor suppressor RAD51C identified by comprehensive analysis of cancer variants.

Mutations in homologous recombination (HR) genes, including BRCA1, BRCA2, and the RAD51 paralog RAD51C, predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.

Distinct functions of three chromatin remodelers in activator binding and preinitiation complex assembly.

The nucleosome remodeling complexes (CRs) SWI/SNF, RSC, and Ino80C cooperate in evicting or repositioning nucleosomes to produce nucleosome depleted regions (NDRs) at the promoters of many yeast genes induced by amino acid starvation. We analyzed mutants depleted of the catalytic subunits of these CRs for binding of transcriptional activator Gcn4 and recruitment of TATA-binding protein (TBP) during preinitiation complex (PIC) assembly. RSC and Ino80 were found to enhance Gcn4 binding to both UAS elements in NDRs upstream of promoters and to unconventional binding sites within nucleosome-occupied coding sequences; and SWI/SNF contributes to UAS binding when RSC is depleted. All three CRs are actively recruited by Gcn4 to most UAS elements and appear to enhance Gcn4 binding by reducing nucleosome occupancies at the binding motifs, indicating a positive regulatory loop. SWI/SNF acts unexpectedly in WT cells to prevent excessive Gcn4 binding at many UAS elements, indicating a dual mode of action that is modulated by the presence of RSC. RSC and SWI/SNF collaborate to enhance TBP recruitment at Gcn4 target genes, together with Ino80C, in a manner associated with nucleosome eviction at the TBP binding sites. Cooperation among the CRs in TBP recruitment is also evident at the highly transcribed ribosomal protein genes, while RSC and Ino80C act more broadly than SWI/SNF at the majority of other constitutively expressed genes to stimulate this step in PIC assembly. Our findings indicate a complex interplay among the CRs in evicting promoter nucleosomes to regulate activator binding and stimulate PIC assembly.

Long-term survival of an ovarian cancer patient harboring a RAD51C missense mutation.

Mutations in homologous recombination (HR) genes predispose to cancer but also sensitize to chemotherapeutics. Although therapy can initially be effective, cancers frequently cease responding, leading to recurrence and poor prognosis. Here we identify a germline mutation in RAD51C, a critical HR factor and known tumor suppressor, in an ovarian cancer patient with exceptionally long, progression-free survival. The RAD51C-T132P mutation is in a highly conserved residue within the nucleotide-binding site and interferes with single-strand DNA binding of the RAD51 paralog complex RAD51B-RAD51C-RAD51D-XRCC2 and association with another RAD51 paralog XRCC3. These biochemical defects lead to highly defective HR and drug sensitivity in tumor cells, ascribing RAD51C-T132P as a deleterious mutation that was likely causal for tumor formation. Conversely, its position within a critical site suggests that it is refractory to secondary mutations that would restore RAD51C gene function and lead to therapy resistance. A need for a greater understanding of the relationship between mutation position and reversion potential of HR genes is underscored, as it may help predict the effectiveness of therapies in patients with HR-deficient cancers.

Chromatin remodeler Ino80C acts independently of H2A.Z to evict promoter nucleosomes and stimulate transcription of highly expressed genes in yeast.

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.

SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast.

The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of nucleosomes-during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.

Gcn4 Binding in Coding Regions Can Activate Internal and Canonical 5' Promoters in Yeast.

Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing ∼30% of Gcn4-binding motifs. Surprisingly, only ∼40% of the bound sites are in promoters, of which only ∼60% activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ∼300 Gcn4-bound sites are within coding sequences (CDSs), with ∼75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDSs adjacent to induced TBP peaks, consistent with Gcn4 activation of cryptic bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDSs can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters. Our results show that internal promoters can be regulated by an activator that functions at conventional 5'-positioned promoters.

Genome-wide cooperation by HAT Gcn5, remodeler SWI/SNF, and chaperone Ydj1 in promoter nucleosome eviction and transcriptional activation.

Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes.

Accumulation of a threonine biosynthetic intermediate attenuates general amino acid control by accelerating degradation of Gcn4 via Pho85 and Cdk8.

Gcn4 is a master transcriptional regulator of amino acid and vitamin biosynthetic enzymes subject to the general amino acid control (GAAC), whose expression is upregulated in response to amino acid starvation in Saccharomyces cerevisiae. We found that accumulation of the threonine pathway intermediate ß-aspartate semialdehyde (ASA), substrate of homoserine dehydrogenase (Hom6), attenuates the GAAC transcriptional response by accelerating degradation of Gcn4, already an exceedingly unstable protein, in cells starved for isoleucine and valine. The reduction in Gcn4 abundance on ASA accumulation requires Cdk8/Srb10 and Pho85, cyclin-dependent kinases (CDKs) known to mediate rapid turnover of Gcn4 by the proteasome via phosphorylation of the Gcn4 activation domain under nonstarvation conditions. Interestingly, rescue of Gcn4 abundance in hom6 cells by elimination of SRB10 is not accompanied by recovery of transcriptional activation, while equivalent rescue of UAS-bound Gcn4 in hom6 pho85 cells restores greater than wild-type activation of Gcn4 target genes. These and other findings suggest that the two CDKs target different populations of Gcn4 on ASA accumulation, with Srb10 clearing mostly inactive Gcn4 molecules at the promoter that are enriched for sumoylation of the activation domain, and Pho85 clearing molecules unbound to the UAS that include both fully functional and inactive Gcn4 species.

Sphingolipid biosynthetic pathway genes FEN1 and SUR4 modulate amphotericin B resistance.

Deletants of the sphingolipid biosynthetic pathway genes FEN1 and SUR4 of Saccharomyces cerevisiae, as well as deletants of their orthologs in Candida albicans, were found to be 2- to 5-fold-more sensitive to amphotericin B (AmB) than parent strains. The inhibition of sphingolipid biosynthesis in parent strains by myriocin sensitized them to AmB, which can be reversed by providing phytosphingosine, an intermediate in the sphingolipid pathway. These results indicate that sphingolipids modulate AmB resistance, with implications for mechanisms underlying AmB action and resistance.

Conserved Ser/Arg-rich motif in PPZ orthologs from fungi is important for its role in cation tolerance.

PPZ1 orthologs, novel members of a phosphoprotein phosphatase family of phosphatases, are found only in fungi. They regulate diverse physiological processes in fungi e.g. ion homeostasis, cell size, cell integrity, etc. Although they are an important determinant of salt tolerance in fungi, their physiological role remained unexplored in any halotolerant species. In this context we report here molecular and functional characterization of DhPPZ1 from Debaryomyces hansenii, which is one of the most halotolerant and osmotolerant species of yeast. Our results showed that DhPPZ1 knock-out strain displayed higher tolerance to toxic cations, and unlike in Saccharomyces cerevisiae, Na(+)/H(+) antiporter appeared to have an important role in this process. Besides salt tolerance, DhPPZ1 also had role in cell wall integrity and growth in D. hansenii. We have also identified a short, serine-arginine-rich sequence motif in DhPpz1p that is essential for its role in salt tolerance but not in other physiological processes. Taken together, these results underscore a distinct role of DhPpz1p in D. hansenii and illustrate an example of how organisms utilize the same molecular tool box differently to garner adaptive fitness for their respective ecological niches.