Dynamic localization of human RAD18 during the cell cycle and a functional connection with DNA double-strand break repair.
The ubiquitin ligase RAD18 is involved in different DNA repair processes. Here, we show that in G1 phase, human RAD18 accumulates in a few relatively large spontaneous foci that contain proteins involved in double-strand break (DSB) repair. These foci persist until cells enter S phase, when numerous small foci appear. At these sites, only 20% of RAD18 colocalizes with PCNA, a known RAD18 substrate. In late G2 phase, RAD18 relocates to nucleoli. After UVC irradiation, PCNA accumulates at the damaged site, followed by RAD18, independent of the cell cycle phase. After induction of DSBs, using low-power multi-photon laser, RAD18 accumulated at the DSB sites, but no PCNA accumulation was observed. Our data show that RAD18 accumulates on DSBs independent of the cell cycle phase. DSBs marked by RAD18 and RAD51 are also positive for RPA in G1 phase, and these DSBs persist until S phase. In addition, we show that DSBs generated in G2 phase are not all repaired, and are observed again in the next G1 phase. We conclude that repair of induced and spontaneous DSBs that accumulate RAD18 and RAD51 in G1 phase cells is delayed until S phase.
Inagaki
Akiko
A
Department of Reproduction and Development, Erasmus MC, University Medical Center, PO Box 2040, 3000 CA, Rotterdam, The Netherlands.
van Cappellen
Wiggert A
WA
van der Laan
Roald
R
Houtsmuller
Adriaan B
AB
Hoeijmakers
Jan H J
JH
Grootegoed
J Anton
JA
Baarends
Willy M
WM
D002453:Cell Cycle / Q000528:radiation effects; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D016193:G1 Phase / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007834:Lasers; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D021381:Protein Transport / Q000528:radiation effects; D011839:Radiation, Ionizing; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase / Q000528:radiation effects; D044767:Ubiquitin-Protein Ligases; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; D011993:Recombinant Fusion Proteins; D044767:Ubiquitin-Protein Ligases