A systematic quantitative approach comprehensively defines domain-specific functional pathways linked to Schizosaccharomyces pombe heterochromatin regulation.

Heterochromatin plays a critical role in regulating gene expression and maintaining genome integrity. While structural and enzymatic components have been linked to heterochromatin establishment, a comprehensive view of the underlying pathways at diverse heterochromatin domains remains elusive. Here, we developed a systematic approach to identify factors involved in heterochromatin silencing at pericentromeres, subtelomeres and the silent mating type locus in Schizosaccharomyces pombe.

SUMO protease and proteasome recruitment at the nuclear periphery differently affect replication dynamics at arrested forks.

Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase.

A marker-free genome editing method in using the approach.

The fission yeast, like budding yeast, offer an easy manipulation of their genome, despite their distinct biology. Most tools available in budding yeast are also available in fission yeast in versions taking into account the features of each organism. The is a powerful approach, initially developed in , for site-directed mutagenesis.

Widely spaced and divergent inverted repeats become a potent source of chromosomal rearrangements in long single-stranded DNA regions.

DNA inverted repeats (IRs) are widespread across many eukaryotic genomes. Their ability to form stable hairpin/cruciform secondary structures is causative in triggering chromosome instability leading to several human diseases. Distance and sequence divergence between IRs are inversely correlated with their ability to induce gross chromosomal rearrangements (GCRs) because of a lesser probability of secondary structure formation and chromosomal breakage.

RNA:DNA hybrids from Okazaki fragments contribute to establish the Ku-mediated barrier to replication-fork degradation.

Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku barrier to nascent strand degradation.

Characterization of canavanine-resistance of cat1 and vhc1 deletions and a dominant any1 mutation in fission yeast.

Positive and counter-selectable markers have been successfully integrated as a part of numerous genetic assays in many model organisms. In this study, we investigate the mechanism of resistance to arginine analog canavanine and its applicability for genetic selection in Schizosaccharomyces pombe. Deletion of both the arginine permease gene cat1 and SPBC18H10.

Structural parameters of palindromic repeats determine the specificity of nuclease attack of secondary structures.

Palindromic sequences are a potent source of chromosomal instability in many organisms and are implicated in the pathogenesis of human diseases. In this study, we investigate which nucleases are responsible for cleavage of the hairpin and cruciform structures and generation of double-strand breaks at inverted repeats in Saccharomyces cerevisiae. We demonstrate that the involvement of structure-specific nucleases in palindrome fragility depends on the distance between inverted repeats and their transcriptional status.

The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal.

Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR).

The Analysis of Recombination-Dependent Processing of Blocked Replication Forks by Bidimensional Gel Electrophoresis.

The perturbation of the DNA replication process is a threat to genome stability and is an underlying cause of cancer development and numerous human diseases. It has become central to understanding how stressed replication forks are processed to avoid their conversion into fragile and pathological DNA structures. The engineering of replication fork barriers (RFBs) to conditionally induce the arrest of a single replisome at a defined locus has made a tremendous impact in our understanding of replication fork processing.

Genetic and Molecular Approaches to Study Chromosomal Breakage at Secondary Structure-Forming Repeats.

DNA repeats capable of adopting stable secondary structures are hotspots for double-strand break (DSB) formation and, hence, for homologous recombination and gross chromosomal rearrangements (GCR) in many prokaryotic and eukaryotic organisms, including humans. Here, we provide protocols for studying chromosomal instability triggered by hairpin- and cruciform-forming palindromic sequences in the budding yeast, Saccharomyces cerevisiae. First, we describe two sensitive genetic assays aimed to determine the recombinogenic potential of inverted repeats and their ability to induce GCRs.

Histone deposition promotes recombination-dependent replication at arrested forks.

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored.

Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks.

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break.

Preserving replication fork integrity and competence via the homologous recombination pathway.

Flaws in the DNA replication process have emerged as a leading driver of genome instability in human diseases. Alteration to replication fork progression is a defining feature of replication stress and the consequent failure to maintain fork integrity and complete genome duplication within a single round of S-phase compromises genetic integrity. This includes increased mutation rates, small and large scale genomic rearrangement and deleterious consequences for the subsequent mitosis that result in the transmission of additional DNA damage to the daughter cells.

The end-joining factor Ku acts in the end-resection of double strand break-free arrested replication forks.

Replication requires homologous recombination (HR) to stabilize and restart terminally arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily double strand breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe.

Unprotected Replication Forks Are Converted into Mitotic Sister Chromatid Bridges.

Replication stress and mitotic abnormalities are key features of cancer cells. Temporarily paused forks are stabilized by the intra-S phase checkpoint and protected by the association of Rad51, which prevents Mre11-dependent resection. However, if a fork becomes dysfunctional and cannot resume, this terminally arrested fork is rescued by a converging fork to avoid unreplicated parental DNA during mitosis.