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Steric gate variants of UmuC confer UV hypersensitivity on Escherichia coli
Brenna W Shurtleff1, Jaylene N Ollivierre, Mohammad Tehrani
1Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
Abstract:
Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contribution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contacts with their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit specificity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in the specific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gate residue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UV light. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UV hypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in the known motifs in UmuC responsible for binding to the beta processivity clamp, suggesting that the UmuC steric gate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant also exhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNA polymerase proofreading subunit suppressed the dominant negative phenotype of a UmuC steric gate variant. This could be due to a recruitment function of the proofreading subunit or involvement of the proofreading subunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.
Insights
Mutations in Y family DNA polymerases UmuC affect DNA repair, causing UV light sensitivity. Disruption of the proofreading subunit suppressed this effect, suggesting a novel interaction in DNA replication and repair.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Y family DNA polymerases are crucial for replicating damaged DNA and confer resistance to DNA lesions.
- Unlike replicative polymerases, Y family polymerases have fewer DNA substrate contacts but exhibit lesion specificity.
- Previous studies showed steric gate mutations in E. coli DinB specifically abolished lesion bypass activity.
Purpose of the Study:
- To investigate the role of steric gate residues in E. coli UmuC function.
- To determine the impact of UmuC steric gate mutations on DNA repair and mutagenesis.
- To elucidate the mechanism by which UmuC steric gate variants affect replication fork access and cellular response to UV light.
Main Methods:
- Construction and characterization of E. coli UmuC variants with mutations at steric gate residues Y11 and F10.
- Assessment of UV light sensitivity and mutagenesis in strains expressing UmuC variants.
- Analysis of dominant-negative effects and suppression by mutations in beta-clamp binding motifs.
- Investigation of the effect of dnaQ gene disruption on UmuC variant phenotypes.
Main Results:
- UmuC variants with Y11 or F10 mutations caused hypersensitivity to UV light and exhibited a dominant-negative phenotype.
- UV hypersensitivity and dominant-negative effects were partially suppressed by mutations in UmuC's beta-clamp binding motifs.
- Strains expressing the UmuC Y11A variant showed decreased UV mutagenesis.
- Disruption of the dnaQ gene (encoding the proofreading subunit) suppressed the dominant-negative phenotype of the UmuC steric gate variant.
Conclusions:
- The steric gate of E. coli UmuC plays a critical role in lesion bypass and tolerance.
- UmuC steric gate variants likely interfere with replication fork progression, potentially through altered access.
- The DNA proofreading subunit (DnaQ) may be involved in regulating UmuC function at the replication fork, possibly through recruitment or a futile cycling mechanism.
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