Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae.
Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2-Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Delta cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair.
Szyjka
Shawn J
SJ
Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089, USA.
Aparicio
Jennifer G
JG
Viggiani
Christopher J
CJ
Knott
Simon
S
Xu
Weihong
W
Tavaré
Simon
S
Aparicio
Oscar M
OM
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D047369:Chromatin Immunoprecipitation; D015825:Chromosomes, Fungal / Q000502:physiology*; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D047468:Immunoprecipitation; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000502:physiology; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D008741:Methyl Methanesulfonate; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C502440:PPH3 protein, S cerevisiae; C491716:PTC2 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D010744:Phosphoric Monoester Hydrolases