Role for DNA polymerase kappa in the processing of N2-N2-guanine interstrand cross-links.
Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N(2)-guanines, similar to cross-links formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was approximately 97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) kappa not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 ' and 3 ' ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol zeta or a pol zeta/Rev1 combination. Because pol kappa was able to bypass these ICLs, biological evidence for a role for pol kappa in tolerating the N(2)-N(2)-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol kappa-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol kappa in the processing of N(2)-N(2)-guanine ICLs.
Minko
Irina G
IG
Center for Research on Occupational and Environmental Toxicology and Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon 97239, USA.
Harbut
Michael B
MB
Kozekov
Ivan D
ID
Kozekova
Albena
A
Jakobs
Petra M
PM
Olson
Susan B
SB
Moses
Robb E
RE
Harris
Thomas M
TM
Rizzo
Carmelo J
CJ
Lloyd
R Stephen
RS
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D002875:Chromosomes / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000737:chemistry; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000502:physiology*; D006147:Guanine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009153:Mutagens; D009841:Oligonucleotides / Q000737:chemistry; D015203:Reproducibility of Results; D000099270:Y-Family DNA Polymerases
D003432:Cross-Linking Reagents; D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D009153:Mutagens; D009841:Oligonucleotides; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases