RAD51D- and FANCG-dependent base substitution mutagenesis at the ATP1A1 locus in mammalian cells

John M. Hinz, Salustra S. Urbin, Larry H. Thompson

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Elaborate processes act at the DNA replication fork to minimize the generation of chromatid discontinuity when lesions are encountered. To prevent collapse of stalled replication forks, mutagenic translesion synthesis (TLS) polymerases are recruited temporarily to bypass DNA lesions. When a replication-associated (one-ended) double-strand break occurs, homologous recombination repair (HRR) can restore chromatid continuity in what has traditionally been regarded as an "error-free" process. Our previous mutagenesis studies show an important role for HRR in preventing deletions and rearrangements that would otherwise result from error-prone nonhomologous end joining (NHEJ) after fork breakage. An analogous, but distinct, role in minimizing mutations is attributed to the proteins defective in the cancer predisposition disease Fanconi anemia (FA). Cells from FA patients and model systems show an increased proportion of gene-disrupting deletions at the hprt locus as well as decreased mutation rates in the hprt assay, suggesting a role for the FANC proteins in promoting TLS, HRR, and possibly also NHEJ. It remains unclear whether HRR, like the FANC pathway, impacts the rate of base substitution mutagenesis. Therefore, we measured, in isogenic rad51d and fancg CHO mutants, mutation rates at the Na+/K+-ATPase α-subunit (ATP1A1) locus using ouabain resistance, which specifically detects base substitution mutations. Surprisingly, we found that the spontaneous mutation rate was reduced ∼2.5-fold in rad51d knockout cells, an even greater extent than observed in fancg cells, when compared with parental and isogenic gene-complemented control lines. A ∼2-fold reduction in induced mutations in rad51d cells was seen after treatment with the DNA alkylating agent ethylnitrosurea while a lesser reduction occurred in fancg cells. Should the model ATP1A1 locus be representative of the genome, we conclude that at least 50% of base substitution mutations in this mammalian system arise through error-prone polymerase(s) acting during HRR-mediated restart of broken replication forks.

Original languageEnglish (US)
Pages (from-to)61-66
Number of pages6
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume665
Issue number1-2
DOIs
StatePublished - Jun 1 2009
Externally publishedYes

Fingerprint

Recombinational DNA Repair
Mutagenesis
Mutation Rate
Fanconi Anemia
Mutation
Chromatids
Fanconi Anemia Complementation Group Proteins
Alkylating Agents
DNA
Gene Deletion
Ouabain
DNA Replication
Genome
Genes
Neoplasms
Proteins

Keywords

  • CHO cells
  • Fanconi anemia
  • Homologous recombination
  • Ouabain resistance
  • Translesion synthesis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Cite this

RAD51D- and FANCG-dependent base substitution mutagenesis at the ATP1A1 locus in mammalian cells. / Hinz, John M.; Urbin, Salustra S.; Thompson, Larry H.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 665, No. 1-2, 01.06.2009, p. 61-66.

Research output: Contribution to journalArticle

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AB - Elaborate processes act at the DNA replication fork to minimize the generation of chromatid discontinuity when lesions are encountered. To prevent collapse of stalled replication forks, mutagenic translesion synthesis (TLS) polymerases are recruited temporarily to bypass DNA lesions. When a replication-associated (one-ended) double-strand break occurs, homologous recombination repair (HRR) can restore chromatid continuity in what has traditionally been regarded as an "error-free" process. Our previous mutagenesis studies show an important role for HRR in preventing deletions and rearrangements that would otherwise result from error-prone nonhomologous end joining (NHEJ) after fork breakage. An analogous, but distinct, role in minimizing mutations is attributed to the proteins defective in the cancer predisposition disease Fanconi anemia (FA). Cells from FA patients and model systems show an increased proportion of gene-disrupting deletions at the hprt locus as well as decreased mutation rates in the hprt assay, suggesting a role for the FANC proteins in promoting TLS, HRR, and possibly also NHEJ. It remains unclear whether HRR, like the FANC pathway, impacts the rate of base substitution mutagenesis. Therefore, we measured, in isogenic rad51d and fancg CHO mutants, mutation rates at the Na+/K+-ATPase α-subunit (ATP1A1) locus using ouabain resistance, which specifically detects base substitution mutations. Surprisingly, we found that the spontaneous mutation rate was reduced ∼2.5-fold in rad51d knockout cells, an even greater extent than observed in fancg cells, when compared with parental and isogenic gene-complemented control lines. A ∼2-fold reduction in induced mutations in rad51d cells was seen after treatment with the DNA alkylating agent ethylnitrosurea while a lesser reduction occurred in fancg cells. Should the model ATP1A1 locus be representative of the genome, we conclude that at least 50% of base substitution mutations in this mammalian system arise through error-prone polymerase(s) acting during HRR-mediated restart of broken replication forks.

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