Replication bypass of N 2-deoxyguanosine interstrand cross-links by human DNA polymerases η and ι

Alex R. Klug, Michael B. Harbut, Robert (Stephen) Lloyd, Irina Minko

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

DNA-interstrand cross-links (ICLs) can be repaired by biochemical pathways requiring DNA polymerases that are capable of translesion DNA synthesis (TLS). The anticipated function of TLS polymerases in these pathways is to insert nucleotides opposite and beyond the linkage site. The outcome of these reactions can be either error-free or mutagenic. TLS-dependent repair of ICLs formed between the exocyclic nitrogens of deoxyguanosines (N 2-dG) can result in low-frequency base substitutions, predominantly G to T transversions. Previously, we demonstrated in vitro that error-free bypass of a model acrolein-mediated N 2-dG ICL can be accomplished by human polymerase (pol) κ, while Rev1 can contribute to this bypass by inserting dC opposite the cross-linked dG. The current study characterized two additional human DNA polymerases, pol η and pol ι, with respect to their potential contributions to either error-free or mutagenic bypass of these lesions. In the presence of individual dNTPs, pol η could insert dA, dG, and dT opposite the cross-linked dG, but incorporation of dC was not apparent. Further primer extension was observed only from the dC and dG 3′ termini, and the amounts of products were low relative to the matched undamaged substrate. Analyses of bypass products beyond the adducted site revealed that dG was present opposite the cross-linked dG in the majority of extended primers, and short deletions were frequently detected. When pol ι was tested for its ability to replicate past this ICL, the correct dC was preferentially incorporated, but no further extension was observed. Under the steady-state conditions, the efficiency of dC incorporation was reduced ∼500-fold relative to the undamaged dG. Thus, in addition to pol κ-catalyzed error-free bypass of N 2-dG ICLs, an alternative, albeit low-efficiency, mechanism may exist. In this pathway, either Rev1 or pol ι could insert dC opposite the lesion, while pol η could perform the subsequent extension.

Original languageEnglish (US)
Pages (from-to)755-762
Number of pages8
JournalChemical Research in Toxicology
Volume25
Issue number3
DOIs
StatePublished - Mar 19 2012

Fingerprint

Deoxyguanosine
DNA-Directed DNA Polymerase
DNA
Acrolein
Nitrogen
Nucleotides
Repair
Substitution reactions
8-nitro-2'-deoxyguanosine
Substrates

ASJC Scopus subject areas

  • Toxicology

Cite this

Replication bypass of N 2-deoxyguanosine interstrand cross-links by human DNA polymerases η and ι. / Klug, Alex R.; Harbut, Michael B.; Lloyd, Robert (Stephen); Minko, Irina.

In: Chemical Research in Toxicology, Vol. 25, No. 3, 19.03.2012, p. 755-762.

Research output: Contribution to journalArticle

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abstract = "DNA-interstrand cross-links (ICLs) can be repaired by biochemical pathways requiring DNA polymerases that are capable of translesion DNA synthesis (TLS). The anticipated function of TLS polymerases in these pathways is to insert nucleotides opposite and beyond the linkage site. The outcome of these reactions can be either error-free or mutagenic. TLS-dependent repair of ICLs formed between the exocyclic nitrogens of deoxyguanosines (N 2-dG) can result in low-frequency base substitutions, predominantly G to T transversions. Previously, we demonstrated in vitro that error-free bypass of a model acrolein-mediated N 2-dG ICL can be accomplished by human polymerase (pol) κ, while Rev1 can contribute to this bypass by inserting dC opposite the cross-linked dG. The current study characterized two additional human DNA polymerases, pol η and pol ι, with respect to their potential contributions to either error-free or mutagenic bypass of these lesions. In the presence of individual dNTPs, pol η could insert dA, dG, and dT opposite the cross-linked dG, but incorporation of dC was not apparent. Further primer extension was observed only from the dC and dG 3′ termini, and the amounts of products were low relative to the matched undamaged substrate. Analyses of bypass products beyond the adducted site revealed that dG was present opposite the cross-linked dG in the majority of extended primers, and short deletions were frequently detected. When pol ι was tested for its ability to replicate past this ICL, the correct dC was preferentially incorporated, but no further extension was observed. Under the steady-state conditions, the efficiency of dC incorporation was reduced ∼500-fold relative to the undamaged dG. Thus, in addition to pol κ-catalyzed error-free bypass of N 2-dG ICLs, an alternative, albeit low-efficiency, mechanism may exist. In this pathway, either Rev1 or pol ι could insert dC opposite the lesion, while pol η could perform the subsequent extension.",
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AB - DNA-interstrand cross-links (ICLs) can be repaired by biochemical pathways requiring DNA polymerases that are capable of translesion DNA synthesis (TLS). The anticipated function of TLS polymerases in these pathways is to insert nucleotides opposite and beyond the linkage site. The outcome of these reactions can be either error-free or mutagenic. TLS-dependent repair of ICLs formed between the exocyclic nitrogens of deoxyguanosines (N 2-dG) can result in low-frequency base substitutions, predominantly G to T transversions. Previously, we demonstrated in vitro that error-free bypass of a model acrolein-mediated N 2-dG ICL can be accomplished by human polymerase (pol) κ, while Rev1 can contribute to this bypass by inserting dC opposite the cross-linked dG. The current study characterized two additional human DNA polymerases, pol η and pol ι, with respect to their potential contributions to either error-free or mutagenic bypass of these lesions. In the presence of individual dNTPs, pol η could insert dA, dG, and dT opposite the cross-linked dG, but incorporation of dC was not apparent. Further primer extension was observed only from the dC and dG 3′ termini, and the amounts of products were low relative to the matched undamaged substrate. Analyses of bypass products beyond the adducted site revealed that dG was present opposite the cross-linked dG in the majority of extended primers, and short deletions were frequently detected. When pol ι was tested for its ability to replicate past this ICL, the correct dC was preferentially incorporated, but no further extension was observed. Under the steady-state conditions, the efficiency of dC incorporation was reduced ∼500-fold relative to the undamaged dG. Thus, in addition to pol κ-catalyzed error-free bypass of N 2-dG ICLs, an alternative, albeit low-efficiency, mechanism may exist. In this pathway, either Rev1 or pol ι could insert dC opposite the lesion, while pol η could perform the subsequent extension.

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