A human DNA polymerase eta complex containing Rad18, Rad6 and Rev1; proteomic analysis and targeting of the complex to the chromatin-bound fraction of cells undergoing replication fork arrest.
DNA polymerase eta (Poleta) is responsible for efficient translesion synthesis (TLS) past cis-syn cyclobutane thymine dimers (TT dimers), the major DNA lesions induced by UV irradiation. Loss of human Poleta leads to xeroderma pigmentosum variant syndrome, clearly indicating that Poleta plays a vital role in preventing skin cancer caused by exposure to sunlight. To further examine Poleta functions and the mechanisms that regulate this important protein, Poleta complexes were purified from HeLa cells over-expressing epitope-tagged Poleta, and polypeptides associated with Poleta, including Rad18, Rad6 and Rev1, were identified by a combination of mass spectrometry and Western blot analysis. The chromatin-bound fractions of cells subjected to UV irradiation, S phase synchronization, or S phase arrest were specifically enriched in such complexes. These results suggest that arrested replication forks strengthen interactions among Poleta, Rad18/Rad6 and Rev1, consistent with the requirement for effective TLS by Poleta at sites of DNA lesions.
Yuasa
Mayumi S
MS
Graduate School of Frontier Biosciences, Osaka University, and SORST, Japan Science and Technology Agency, 1-3 Yamada-Oka, Suita, Osaka 565-0871, Japan.
Masutani
Chikahide
C
Hirano
Akihiko
A
Cohn
Martin A
MA
Yamaizumi
Masaru
M
Nakatani
Yoshihiro
Y
Hanaoka
Fumio
F
D002843:Chromatin / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004261:DNA Replication / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases / Q000378:metabolism*; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D044767:Ubiquitin-Protein Ligases; D000099270:Y-Family DNA Polymerases
D002843:Chromatin; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D044767:Ubiquitin-Protein Ligases; C485954:RAD18 protein, human; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein