title: Replication fork collapse and genome instability in a deoxycytidylate deaminase mutant.
abstract: Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1(+) dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Arancha
authors.initials: A
authors.lastname: Sánchez
authors.forename: Sushma
authors.initials: S
authors.lastname: Sharma
authors.forename: Sophie
authors.initials: S
authors.lastname: Rozenzhak
authors.forename: Assen
authors.initials: A
authors.lastname: Roguev
authors.forename: Nevan J
authors.initials: NJ
authors.lastname: Krogan
authors.forename: Andrei
authors.initials: A
authors.lastname: Chabes
authors.forename: Paul
authors.initials: P
authors.lastname: Russell
mesh_terms: D002453:Cell Cycle / Q000235:genetics; D000071877:Checkpoint Kinase 1; D003844:DCMP Deaminase / Q000172:deficiency / Q000235:genetics*; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D003845:Deoxycytosine Nucleotides / Q000096:biosynthesis; D042822:Genomic Instability; D009713:Nucleotidyltransferases / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D013942:Thymine Nucleotides / Q000096:biosynthesis
chemical_list: C511286:Brc1 protein, S pombe; D003845:Deoxycytosine Nucleotides; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D009713:Nucleotidyltransferases; C022348:phosphatidate cytidylyltransferase; D003844:DCMP Deaminase; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases; C024157:thymidine 5'-triphosphate