title: Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.
abstract: The yeast checkpoint kinases Mec1 and Rad53 are required for genomic stability in the presence of replicative stress. When replication forks stall, the stable maintenance of replisome components requires the ATR kinase Mec1/Ddc2 and the RecQ helicase Sgs1. It was unclear whether either Mec1 or Sgs1 action requires the checkpoint effector kinase, Rad53. By combining sgs1Delta with checkpoint-deficient alleles, we can now distinguish the role of Mec1 at stalled forks from that of Rad53. We show that the S-phase-specific mec1-100 allele, like the sgs1Delta mutation, partially destabilizes DNA polymerases at stalled forks, yet combining the mec1-100 and sgs1Delta mutations leads to complete disassociation of the replisome, loss of RPA, irreversible termination of nucleotide incorporation, and compromised recovery from hydroxyurea (HU) arrest. These events coincide with a dramatic increase in both spontaneous and HU-induced chromosomal rearrangements. Importantly, in sgs1Delta cells, RPA levels at stalled forks do not change, although Ddc2 recruitment is compromised, explaining the partial Sgs1 and Mec1 interdependence. Loss of Rad53 kinase, on the other hand, does not affect the levels of DNA polymerases at arrested forks, but leads to MCM protein dissociation. Finally, confirming its unique role during replicative stress, Mec1, and not Tel1, is shown to modify fork-associated histone H2A.
authors.affiliation: Frontiers in Genetics NCCR Program, University of Geneva, Switzerland.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Cobb
authors.forename: Thomas
authors.initials: T
authors.lastname: Schleker
authors.forename: Vanesa
authors.initials: V
authors.lastname: Rojas
authors.forename: Lotte
authors.initials: L
authors.lastname: Bjergbaek
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Tercero
authors.forename: Susan M
authors.initials: SM
authors.lastname: Gasser
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D053484:RecQ Helicases; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014178:Translocation, Genetic
chemical_list: D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C513623:TEL1 protein, S cerevisiae; C067022:RAD53 protein, S cerevisiae; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; D053484:RecQ Helicases