title: Sequential assembly of translesion DNA polymerases at UV-induced DNA damage sites.
abstract: In response to DNA damage such as from UV irradiation, mammalian Y-family translesion synthesis (TLS) polymerases Polη and Rev1 colocalize with proliferating cell nuclear antigen at nuclear foci, presumably representing stalled replication sites. However, it is unclear whether the localization of one polymerase is dependent on another. Furthermore, there is no report on the in vivo characterization of the Rev3 catalytic subunit of the B-family TLS polymerase Polζ. Here we describe the detection of endogenous human Polη, Rev1, and Rev3 by immunocytochemistry using existing or newly created antibodies, as well as various means of inhibiting their expression, which allows us to examine the dynamics of endogenous TLS polymerases in response to UV irradiation. It is found that Rev1 and Polη are independently recruited to the nuclear foci, whereas the Rev3 nuclear focus formation requires Rev1 but not Polη. In contrast, neither Rev1 nor Polη recruitment requires Rev3. To further support these conclusions, we find that simultaneous suppression of Polη and Rev3 results in an additive cellular sensitivity to UV irradiation. These observations suggest a cooperative and sequential assembly of TLS polymerases in response to DNA damage. They also support and extend the current polymerase switch model.
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, S7N 5E5, Canada.
authors.forename: Parker L
authors.initials: PL
authors.lastname: Andersen
authors.forename: Fang
authors.initials: F
authors.lastname: Xu
authors.forename: Barry
authors.initials: B
authors.lastname: Ziola
authors.forename: W Glen
authors.initials: WG
authors.lastname: McGregor
authors.forename: Wei
authors.initials: W
authors.lastname: Xiao
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004273:DNA, Neoplasm / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D045325:HCT116 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
chemical_list: D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D018809:Proliferating Cell Nuclear Antigen; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases; C118285:REV3L protein, human; C112338:Rad30 protein