Role for DNA polymerase κ in the processing of N2-N 2-guanine interstrand cross-links

Irina Minko, Michael B. Harbut, Ivan D. Kozekov, Albena Kozekova, Petra M. Jakobs, Susan Olson, Robb Moses, Thomas M. Harris, Carmelo J. Rizzo, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

104 Citations (Scopus)

Abstract

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 N2-guanines, similar to crosslinks formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was ∼97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) κ 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 ζ or a polζ/Rev1 combination. Because pol κ was able to bypass these ICLs, biological evidence for a role for pol κ in tolerating the N2-N2-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol κ-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol κ in the processing of N2-N2-guanine ICLs.

Original languageEnglish (US)
Pages (from-to)17075-17082
Number of pages8
JournalJournal of Biological Chemistry
Volume283
Issue number25
DOIs
StatePublished - Jun 20 2008

Fingerprint

Guanine
DNA-Directed DNA Polymerase
Processing
Oligodeoxyribonucleotides
Mitomycin
Repair
Cells
Chromosomal Instability
Homologous Recombination
DNA Replication
Yeast
Cell Survival
Yeasts
DNA

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Role for DNA polymerase κ in the processing of N2-N 2-guanine interstrand cross-links. / Minko, Irina; Harbut, Michael B.; Kozekov, Ivan D.; Kozekova, Albena; Jakobs, Petra M.; Olson, Susan; Moses, Robb; Harris, Thomas M.; Rizzo, Carmelo J.; Lloyd, Robert (Stephen).

In: Journal of Biological Chemistry, Vol. 283, No. 25, 20.06.2008, p. 17075-17082.

Research output: Contribution to journalArticle

Minko, I, Harbut, MB, Kozekov, ID, Kozekova, A, Jakobs, PM, Olson, S, Moses, R, Harris, TM, Rizzo, CJ & Lloyd, RS 2008, 'Role for DNA polymerase κ in the processing of N2-N 2-guanine interstrand cross-links', Journal of Biological Chemistry, vol. 283, no. 25, pp. 17075-17082. https://doi.org/10.1074/jbc.M801238200
Minko, Irina ; Harbut, Michael B. ; Kozekov, Ivan D. ; Kozekova, Albena ; Jakobs, Petra M. ; Olson, Susan ; Moses, Robb ; Harris, Thomas M. ; Rizzo, Carmelo J. ; Lloyd, Robert (Stephen). / Role for DNA polymerase κ in the processing of N2-N 2-guanine interstrand cross-links. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 25. pp. 17075-17082.
@article{7939a75cb3a04a7cb58f10b3adb4af80,
title = "Role for DNA polymerase κ in the processing of N2-N 2-guanine interstrand cross-links",
abstract = "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 N2-guanines, similar to crosslinks formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was ∼97{\%} accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) κ 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 ζ or a polζ/Rev1 combination. Because pol κ was able to bypass these ICLs, biological evidence for a role for pol κ in tolerating the N2-N2-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol κ-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol κ in the processing of N2-N2-guanine ICLs.",
author = "Irina Minko and Harbut, {Michael B.} and Kozekov, {Ivan D.} and Albena Kozekova and Jakobs, {Petra M.} and Susan Olson and Robb Moses and Harris, {Thomas M.} and Rizzo, {Carmelo J.} and Lloyd, {Robert (Stephen)}",
year = "2008",
month = "6",
day = "20",
doi = "10.1074/jbc.M801238200",
language = "English (US)",
volume = "283",
pages = "17075--17082",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "25",

}

TY - JOUR

T1 - Role for DNA polymerase κ in the processing of N2-N 2-guanine interstrand cross-links

AU - Minko, Irina

AU - Harbut, Michael B.

AU - Kozekov, Ivan D.

AU - Kozekova, Albena

AU - Jakobs, Petra M.

AU - Olson, Susan

AU - Moses, Robb

AU - Harris, Thomas M.

AU - Rizzo, Carmelo J.

AU - Lloyd, Robert (Stephen)

PY - 2008/6/20

Y1 - 2008/6/20

N2 - 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 N2-guanines, similar to crosslinks formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was ∼97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) κ 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 ζ or a polζ/Rev1 combination. Because pol κ was able to bypass these ICLs, biological evidence for a role for pol κ in tolerating the N2-N2-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol κ-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol κ in the processing of N2-N2-guanine ICLs.

AB - 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 N2-guanines, similar to crosslinks formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was ∼97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) κ 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 ζ or a polζ/Rev1 combination. Because pol κ was able to bypass these ICLs, biological evidence for a role for pol κ in tolerating the N2-N2-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol κ-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol κ in the processing of N2-N2-guanine ICLs.

UR - http://www.scopus.com/inward/record.url?scp=47749128072&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=47749128072&partnerID=8YFLogxK

U2 - 10.1074/jbc.M801238200

DO - 10.1074/jbc.M801238200

M3 - Article

C2 - 18434313

AN - SCOPUS:47749128072

VL - 283

SP - 17075

EP - 17082

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 25

ER -