Chk1 Inhibits E2F6 Repressor Function in Response to Replication Stress to Maintain Cell-Cycle Transcription

1. Download : Download high-res image (184KB)
2. Download : Download full-size image

     

Sen1 Is Recruited to Replication Forks via Ctf4 and Mrc1 and Promotes Genome Stability

1. Download : Download high-res image (161KB)
2. Download : Download full-size image

     

Wss1 Promotes Replication Stress Tolerance by Degrading Histones

1. Download : Download high-res image (98KB)
2. Download : Download full-size image

     

Polyubiquitinated PCNA Recruits the ZRANB3 Translocase to Maintain Genomic Integrity after Replication Stress

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in?vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA?structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.     

TORC2 Is Required to Maintain Genome Stability during S Phase in Fission Yeast

DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair, and reentry into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress but are insensitive to ionizing radiation. We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for the return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication restart following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development.     

Novel Checkpoint Pathway Organization Promotes Genome Stability in Stationary-Phase Yeast Cells

Most DNA alterations occur during DNA replication in the S phase of the cell cycle. However, the majority of eukaryotic cells exist in a nondividing, quiescent state. Little is known about the factors involved in preventing DNA instability within this stationary-phase cell population. Previously, we utilized a unique assay system to identify mutations that increased minisatellite alterations specifically in quiescent cells in Saccharomyces cerevisiae. Here we conducted a modified version of synthetic genetic array analysis to determine if checkpoint signaling components play a role in stabilizing minisatellites in stationary-phase yeast cells. Our results revealed that a subset of checkpoint components, specifically MRC1, CSM3, TOF1, DDC1, RAD17, MEC3, TEL1, MEC1, and RAD53, prevent stationary-phase minisatellite alterations within the quiescent cell subpopulation of stationary-phase cells. Pathway analysis revealed at least three pathways, with MRC1, CSM3, and TOF1 acting in a pathway independent of MEC1 and RAD53. Overall, our data indicate that some well-characterized checkpoint components maintain minisatellite stability in stationary-phase cells but are regulated differently in those cells than in actively growing cells. For the MRC1-dependent pathway, the checkpoint itself may not be the important element; rather, it may be loss of the checkpoint proteins'' other functions that contributes to DNA instability.     

Cyclin Proteolysis and CDK Inhibitors: Two Redundant Pathways to Maintain Genome Stability in Mammalian Cells

Cyclin-dependent kinases (CDKs) are regulated by cyclin proteolysis and CDK inhibitors (CKIs) during mitotic exit and G1 phase in yeast and Drosophila, and disruption of both regulatory pathways leads to genomic instability. Our study using mouse cell lines that constitutively express a stabilized mutant of cyclin A revealed that three CKIs, p21, p27, and Rb-related p107, are responsible for cyclin proteolysis-independent inactivation of CDK during mitotic exit and G1. Enforced expression of cyclin A in the cells lacking all three CKIs induced rapid tetraploidization. Thus, the redundant pathways consisting of cyclin proteolysis and CKIs control CDK activity during mitotic exit and contribute to maintenance of genome stability in mammalian cells.