Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination.
The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase eta (poleta) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that poleta does not form nuclear foci in RAD18(-/-) cells after UV irradiation. Both Rad18 and Rad6 are required for poleta focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with poleta constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, poleta interacts preferentially with monoubiquitinated PCNA, but poldelta does not. These results suggest that Rad18 is crucial for recruitment of poleta to the damaged site through protein-protein interaction and PCNA monoubiquitination.
Watanabe
Kenji
K
Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.
Tateishi
Satoshi
S
Kawasuji
Michio
M
Tsurimoto
Toshiki
T
Inoue
Hirokazu
H
Yamaizumi
Masaru
M
D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects; D006224:Cricetinae; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000502:physiology; D044767:Ubiquitin-Protein Ligases; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D004258:DNA Polymerase III; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D018809:Proliferating Cell Nuclear Antigen; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C485954:RAD18 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases