Edgar Hartsuiker

Novel functional requirements for non‐homologous DNA end joining in Schizosaccharomyces pombe

DNA double strand break (DSB) repair by non‐homologous end joining (NHEJ) in mammalian cells requires the Ku70–Ku80 heterodimer, the DNA‐PK catalytic subunit DNA‐PKcs, as well as DNA ligase IV and Xrcc4. NHEJ of plasmid DSBs in Saccharomyces cerevisiae requires Ku, Xrcc4 and DNA ligase IV, as well as Mre11, Rad50, Xrs2 and DNA damage checkpoint proteins. Saccharomyces cerevisiae Ku is also required for telomere length maintenance and transcriptional silencing. We have characterized NHEJ in Schizosaccharomyces pombe using an extrachromosomal assay and find that, as anticipated, it is Ku70 and DNA ligase IV dependent. Unexpectedly, we find that Rad32, Rad50 (the S.pombe homologues of Mre11 and Rad50, respectively) and checkpoint proteins are not required for NHEJ. Furthermore, although S.pombe Ku70 is required for maintenance of telomere length, it is dispensable for transcriptional silencing at telomeres and is located throughout the nucleus rather than concentrated at the telomeres. Together, these results provide insight into the mechanism of NHEJ and contrast significantly with recent studies in S.cerevisiae.

Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot

Histone acetyltransferases (HATs) and ATP‐dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6‐M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)‐like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1·Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26‐ and Atf1‐dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2‐ADCR homologue) deletion and snf22+gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site‐specific manner.

A supramodular FHA/BRCT-Repeat architecture mediates Nbs1 adaptor function in response to DNA damage

The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.

Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair

To study the role of Rad50 in the DNA damage response, we cloned and deleted the Schizosaccharo myces pombe RAD50 homologue. The deletion is sensitive to a range of DNA‐damaging agents and shows dynamic epistatic interactions with other recombination–repair genes. We show that Rad50 is necessary for recombinational repair of the DNA lesion at the mating‐type locus and that rad50Δ shows slow DNA replication. We also find that Rad50 is not required for slowing down S phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment. Interestingly, in rad50Δ cells, the recombination frequency between two homologous chromosomes is increased at the expense of sister chromatid recombination. We propose that Rad50, an SMC‐like protein, promotes the use of the sister chromatid as the template for homologous recombinational repair. In support of this, we found that Rad50 functions in the same pathway for the repair of MMS‐induced damage as Rad21, the homologue of the Saccharomyces cerevisiae Scc1 cohesin protein. We speculate that Rad50 interacts with the cohesin complex during S phase to assist repair and possibly re‐initiation of replication after replication fork collapse.

Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway

The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood. Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast, Schizosaccharomyces pombe, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways. The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase ɛ) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition). These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.

Ctp1CtIP and Rad32Mre11 Nuclease Activity Are Required for Rec12Spo11 Removal, but Rec12Spo11 Removal Is Dispensable for Other MRN-Dependent Meiotic Functions

ABSTRACT The evolutionarily conserved Mre11/Rad50/Nbs1 (MRN) complex is involved in various aspects of meiosis. Whereas available evidence suggests that the Mre11 nuclease activity might be responsible for Spo11 removal in Saccharomyces cerevisiae, this has not been confirmed experimentally. This study demonstrates for the first time that Mre11 (Schizosaccharomyces pombe Rad32Mre11) nuclease activity is required for the removal of Rec12Spo11. Furthermore, we show that the CtIP homologue Ctp1 is required for Rec12Spo11 removal, confirming functional conservation between Ctp1CtIP and the more distantly related Sae2 protein from Saccharomyces cerevisiae. Finally, we show that the MRN complex is required for meiotic recombination, chromatin remodeling at the ade6-M26 recombination hot spot, and formation of linear elements (which are the equivalent of the synaptonemal complex found in other eukaryotes) but that all of these functions are proficient in a rad50S mutant, which is deficient for Rec12Spo11 removal. These observations suggest that the conserved role of the MRN complex in these meiotic functions is independent of Rec12Spo11 removal.

Topoisomerase I regulates open chromatin and controls gene expression in vivo

DNA topoisomerases regulate the topological state of the DNA double helix and are key enzymes in the processes of DNA replication, transcription and genome stability. Using the fission yeast model Schizosaccharomyces pombe, we investigate genome wide how DNA topoisomerases I and II affect chromatin dynamics and gene expression in vivo. We show that topoisomerase I activity is directly required for efficient nucleosome disassembly at gene promoter regions. Lack of topoisomerase activity results in increased nucleosome occupancy, perturbed histone modifications and reduced transcription from these promoters. Strong correlative evidence suggests that topoisomerase I cooperates with the ATP‐dependent chromatin remodeller Hrp1 in nucleosome disassembly. Our study links topoisomerase activity to the maintenance of open chromatin and regulating transcription in vivo.

The msh2 Gene of Schizosaccharomyces pombe Is Involved in Mismatch Repair, Mating-Type Switching, and Meiotic Chromosome Organization

ABSTRACT We have identified in the fission yeast Schizosaccharomyces pombe a MutS homolog that shows highest homology to the Msh2 subgroup. msh2 disruption gives rise to increased mitotic mutation rates and increased levels of postmeiotic segregation of genetic markers. In bandshift assays performed with msh2Δ cell extracts, a general mismatch-binding activity is absent. By complementation assays, we showed that S. pombe msh2 is allelic with the previously identified swi8 andmut3 genes, which are involved in mating-type switching. The swi8-137 mutant has a mutation in the msh2gene which causes a truncated Msh2 peptide lacking a putative DNA-binding domain. Cytological analysis revealed that during meiotic prophase of msh2-defective cells, chromosomal structures were frequently formed; such structures are rarely found in the wild type. Our data show that besides having a function in mismatch repair,S. pombe msh2 is required for correct termination of copy synthesis during mating-type switching as well as for proper organization of chromosomes during meiosis.

Double-strand break repair and homologous recombination in Schizosaccharomyces pombe

The study of double‐strand break repair and homologous recombination in Saccharomyces cerevisiae meiosis has provided important information about the mechanisms involved. However, it has become clear that the resulting recombination models are only partially applicable to repair in mitotic cells, where crossover formation is suppressed. In recent years our understanding of double‐strand break repair and homologous recombination in Schizosaccharomyces pombe has increased significantly, and the identification of novel pathways and genes with homologues in higher eukaryotes has increased its value as a model organism for double‐strand break repair. In this review we will focus on the involvement of homologous recombination and repair in different aspects of genome stability in Sz. pombe meiosis, replication and telomere maintenance. We will also discuss anti‐recombination pathways (that suppress crossover formation), non‐homologous end‐joining, single‐strand annealing and factors that influence the choice and prevalence of the different repair pathways in Sz. pombe. Copyright