The 3′ → 5′ Exonuclease of T4 DNA Polymerase Removes Premutagenic Alkyl Mispairs and Contributes to Futile Cycling at O 6-Methylguanine Lesions

Vineeta Khare, Kristin Eckert

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

We have studied the processing of O6-methylguanine (m6G)-containing oligonucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3′ → 5′ exonuclease of T4 DNA polymerase. In vitro biochemical analyses demonstrate that the exonuclease can remove bases opposite a defined m6G lesion. The efficiency of excision of a terminal m6G·T was similar to that of m6G·C, and both were excised as efficiently as a G·T substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3′ → 5′ exonuclease may contribute to DNA synthesis inhibition by alkylating agents. Genetic data obtained using an in vitro herpes simplex virus-thymidine kinase assay support the inefficiency of the exonuclease as a "proofreading" activity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G → A transitions. Comparison of MNU dose-response curves for exonuclease-proficient and -deficient forms of T4 polymerase reveals that the exonuclease efficiently removes 50-86% of total premutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the biochemical explanation for cellular cytotoxicity of methylating agents.

Original languageEnglish (US)
Pages (from-to)24286-24292
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number26
DOIs
StatePublished - Jun 29 2001

Fingerprint

Substrate Cycling
Exonucleases
DNA-Directed DNA Polymerase
Methylnitrosourea
Assays
DNA
Thymidine Kinase
O-(6)-methylguanine
Alkylating Agents
Substrates
Simplexvirus
Cytotoxicity
Viruses
Oligonucleotides
DNA Repair
Repair
Mutation

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{f1e30c5f13224e349fb725630a1c5ce6,
title = "The 3′ → 5′ Exonuclease of T4 DNA Polymerase Removes Premutagenic Alkyl Mispairs and Contributes to Futile Cycling at O 6-Methylguanine Lesions",
abstract = "We have studied the processing of O6-methylguanine (m6G)-containing oligonucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3′ → 5′ exonuclease of T4 DNA polymerase. In vitro biochemical analyses demonstrate that the exonuclease can remove bases opposite a defined m6G lesion. The efficiency of excision of a terminal m6G·T was similar to that of m6G·C, and both were excised as efficiently as a G·T substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3′ → 5′ exonuclease may contribute to DNA synthesis inhibition by alkylating agents. Genetic data obtained using an in vitro herpes simplex virus-thymidine kinase assay support the inefficiency of the exonuclease as a {"}proofreading{"} activity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G → A transitions. Comparison of MNU dose-response curves for exonuclease-proficient and -deficient forms of T4 polymerase reveals that the exonuclease efficiently removes 50-86{\%} of total premutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the biochemical explanation for cellular cytotoxicity of methylating agents.",
author = "Vineeta Khare and Kristin Eckert",
year = "2001",
month = "6",
day = "29",
doi = "10.1074/jbc.M011025200",
language = "English (US)",
volume = "276",
pages = "24286--24292",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "26",

}

TY - JOUR

T1 - The 3′ → 5′ Exonuclease of T4 DNA Polymerase Removes Premutagenic Alkyl Mispairs and Contributes to Futile Cycling at O 6-Methylguanine Lesions

AU - Khare, Vineeta

AU - Eckert, Kristin

PY - 2001/6/29

Y1 - 2001/6/29

N2 - We have studied the processing of O6-methylguanine (m6G)-containing oligonucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3′ → 5′ exonuclease of T4 DNA polymerase. In vitro biochemical analyses demonstrate that the exonuclease can remove bases opposite a defined m6G lesion. The efficiency of excision of a terminal m6G·T was similar to that of m6G·C, and both were excised as efficiently as a G·T substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3′ → 5′ exonuclease may contribute to DNA synthesis inhibition by alkylating agents. Genetic data obtained using an in vitro herpes simplex virus-thymidine kinase assay support the inefficiency of the exonuclease as a "proofreading" activity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G → A transitions. Comparison of MNU dose-response curves for exonuclease-proficient and -deficient forms of T4 polymerase reveals that the exonuclease efficiently removes 50-86% of total premutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the biochemical explanation for cellular cytotoxicity of methylating agents.

AB - We have studied the processing of O6-methylguanine (m6G)-containing oligonucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3′ → 5′ exonuclease of T4 DNA polymerase. In vitro biochemical analyses demonstrate that the exonuclease can remove bases opposite a defined m6G lesion. The efficiency of excision of a terminal m6G·T was similar to that of m6G·C, and both were excised as efficiently as a G·T substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3′ → 5′ exonuclease may contribute to DNA synthesis inhibition by alkylating agents. Genetic data obtained using an in vitro herpes simplex virus-thymidine kinase assay support the inefficiency of the exonuclease as a "proofreading" activity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G → A transitions. Comparison of MNU dose-response curves for exonuclease-proficient and -deficient forms of T4 polymerase reveals that the exonuclease efficiently removes 50-86% of total premutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the biochemical explanation for cellular cytotoxicity of methylating agents.

UR - http://www.scopus.com/inward/record.url?scp=0035968340&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035968340&partnerID=8YFLogxK

U2 - 10.1074/jbc.M011025200

DO - 10.1074/jbc.M011025200

M3 - Article

C2 - 11290737

AN - SCOPUS:0035968340

VL - 276

SP - 24286

EP - 24292

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 26

ER -