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Error rate and specificity of human and murine DNA polymerase eta
T Matsuda1, K Bebenek, C Masutani
1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Abstract:
We describe here the error specificity of mammalian DNA polymerase eta (pol eta), an enzyme that performs translesion DNA synthesis and may participate in somatic hypermutation of immunoglobulin genes. Both mouse and human pol eta lack intrinsic proofreading exonuclease activity and both copy undamaged DNA inaccurately. Analysis of more than 1500 single-base substitutions by human pol eta indicates that error rates for all 12 mismatches are high and variable depending on the composition and symmetry of the mismatch and its location. pol eta also generates tandem base substitutions at an unprecedented rate, and kinetic analysis indicates that it extends a tandem double mismatch about as efficiently as other replicative enzymes extend single-base mismatches. This ability to use an aberrant primer terminus and the high rate of single and double-base substitutions support the idea that pol eta may forego strict shape complementarity in order to facilitate highly efficient lesion bypass. Relaxed discrimination is further indicated by pol eta infidelity for a wide variety of nucleotide deletion and addition errors. The nature and location of these errors suggest that some may be initiated by strand slippage, while others result from additional mechanisms.
Insights
Mammalian DNA polymerase eta (pol eta) exhibits high error rates during DNA synthesis, generating numerous single-base and tandem substitutions. This enzyme
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA polymerase eta (pol eta) is involved in translesion DNA synthesis and immunoglobulin gene hypermutation.
- Mammalian pol eta lacks proofreading exonuclease activity and exhibits infidelity on undamaged DNA.
Purpose of the Study:
- To characterize the error specificity of mammalian DNA polymerase eta.
- To investigate the mechanisms underlying pol eta's infidelity during DNA replication.
Main Methods:
- Analysis of over 1500 single-base substitutions by human pol eta.
- Kinetic analysis of tandem base substitutions.
- Evaluation of nucleotide deletion and addition errors.
Main Results:
- Human pol eta displays high and variable error rates for all 12 mismatches, influenced by mismatch characteristics and location.
- Pol eta generates tandem base substitutions at an exceptionally high rate, efficiently extending aberrant primer termini.
- The enzyme exhibits relaxed discrimination, leading to frequent nucleotide deletions and additions, potentially via strand slippage.
Conclusions:
- Pol eta's error profile suggests a trade-off between lesion bypass efficiency and replication fidelity.
- The enzyme's ability to utilize aberrant primer termini and its high substitution rate support its role in facilitating lesion bypass.
- Relaxed discrimination contributes to pol eta's infidelity, with errors potentially arising from strand slippage and other mechanisms.