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Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro
Dekang Liu1,2, Jane H Frederiksen1,2, Sascha E Liberti1,2
1Center for Healthy Aging, University of Copenhagen, Denmark.
Abstract:
DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display a mild mutator phenotype, while nuclear extracts of these cells exhibit reduced MMR activity in vitro, and these defects are complemented by overexpression or addition of exogenous human Exonuclease 1 (EXO1). By contrast, another proofreading-deficient mutant, PolδD515V, which has a weaker strand displacement activity, does not decrease the MMR activity as significantly as PolδD316A;E318A. In addition, PolδD515V does not increase the mutation frequency in MMR-proficient cells. Based on our findings, we propose that the proofreading activity restricts the strand displacement activity of Polδ in MMR. This contributes to maintain the nicks required for EXO1 entry, and in this manner ensures the dominance of the EXO1-dependent MMR pathway.
Insights
DNA mismatch repair (MMR) prevents mutations by correcting replication errors. Proofreading-deficient DNA polymerase delta impairs MMR, increasing cancer risk, but Exonuclease 1 can restore repair function.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair Mechanisms
Background:
- DNA mismatch repair (MMR) is crucial for genome stability and preventing cancer.
- Defects in MMR are linked to increased cancer susceptibility.
- Human replicative DNA polymerase delta (Polδ) plays a role in DNA replication and repair.
Purpose of the Study:
- To investigate the interaction between DNA mismatch repair (MMR) and proofreading-deficient alleles of human DNA polymerase delta (Polδ).
- To determine how Polδ's proofreading deficiency affects MMR activity and mutation frequency.
- To elucidate the role of Exonuclease 1 (EXO1) in MMR when Polδ proofreading is compromised.
Main Methods:
- Characterization of human cell lines overexpressing specific proofreading-deficient Polδ mutants (PolδD316A;E318A and PolδD515V).
- Assessment of MMR activity in nuclear extracts using in vitro assays.
- Evaluation of mutation frequency in MMR-proficient cells.
- Complementation assays involving overexpression or addition of exogenous human Exonuclease 1 (EXO1).
Main Results:
- Overexpression of PolδD316A;E318A, a mutant with enhanced strand displacement activity, led to a mild mutator phenotype and reduced MMR activity in vitro.
- The reduced MMR activity in cells overexpressing PolδD316A;E318A was complemented by EXO1.
- A different proofreading-deficient mutant, PolδD515V, with weaker strand displacement activity, caused less significant reductions in MMR activity and did not substantially increase mutation frequency.
- Findings suggest Polδ proofreading activity normally restricts its strand displacement, maintaining nicks essential for EXO1-dependent MMR.
Conclusions:
- Proofreading-deficient Polδ alleles can impair DNA mismatch repair (MMR) efficiency.
- The strand displacement activity of Polδ is implicated in MMR pathway regulation.
- Exonuclease 1 (EXO1) plays a critical role in compensating for impaired MMR due to Polδ proofreading defects, ensuring genome stability.