PTIP/Swift is required for efficient PCNA ubiquitination in response to DNA damage.
Monoubiquitination of proliferating cell nuclear antigen (PCNA) enables translesion synthesis (TLS) by specialized DNA polymerases to replicate past damaged DNA. We have studied PCNA modification and chromatin recruitment of TLS polymerases in Xenopus egg extracts and mammalian cells. We show that Xenopus PCNA becomes ubiquitinated and sumoylated after replication stress induced by UV or aphidicolin. Under these conditions the TLS polymerase eta was recruited to chromatin and also became monoubiquitinated. PTIP/Swift is an adaptor protein for the ATM/ATR kinases. Immunodepletion of PTIP/Swift from Xenopus extracts prevented efficient PCNA ubiquitination and polymerase eta recruitment to chromatin during replicative stress. In addition to PCNA ubiquitination, efficient polymerase eta recruitment to chromatin also required ATR kinase activity. We also show that PTIP depletion from mammalian cells by RNAi reduced PCNA ubiquitination in response to DNA damage, and also decreased the recruitment to chromatin of polymerase eta and the recombination protein Rad51. Our results suggest that PTIP/Swift is an important new regulator of DNA damage avoidance in metazoans.
Göhler
Thomas
T
College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Munoz
Ivan M
IM
Rouse
John
J
Blow
J Julian
JJ
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000528:radiation effects; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D013094:Spermatozoa / Q000187:drug effects / Q000528:radiation effects; D015534:Trans-Activators / Q000378:metabolism*; D054875:Ubiquitination; D014466:Ultraviolet Rays; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*
D002352:Carrier Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; C429305:PAXIP1 protein, Xenopus; C485876:PAXIP1 protein, human; D018809:Proliferating Cell Nuclear Antigen; D015534:Trans-Activators; D029867:Xenopus Proteins; D016590:Aphidicolin