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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Published on: June 19, 2018

Proofreading of DNA polymerase eta-dependent replication errors

K Bebenek1, T Matsuda, C Masutani

  • 1Laboratory of Molecular Genetics and Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

The Journal of Biological Chemistry
|December 13, 2000
PubMed

Insights

Human DNA polymerase eta bypasses UV DNA damage but has low fidelity. Subsequent proofreading by extrinsic exonucleases corrects errors, reducing UV-induced mutagenesis and skin cancer risk.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Human DNA polymerase eta (Pol eta) is encoded by the XPV gene, linked to skin cancer susceptibility.
  • Pol eta bypasses UV photoproducts, a function crucial for DNA replication but potentially mutagenic due to low fidelity.
  • Pol eta lacks intrinsic proofreading, raising questions about error correction mechanisms.

Purpose of the Study:

  • To investigate the fidelity and error correction mechanisms of human DNA polymerase eta during DNA replication.
  • To determine if Pol eta's low fidelity errors can be corrected by extrinsic proofreading systems.
  • To understand how Pol eta balances efficient UV lesion bypass with minimizing mutagenic potential.

Main Methods:

  • Replication assays using human cell extracts.
  • Competition experiments between Pol eta and other human DNA polymerases.
  • Analysis of Pol eta's processivity and primer extension efficiency at matched and mismatched termini.
  • Investigating the effect of dNTP concentration and specific nucleotide addition (dGMP) on Pol eta-induced infidelity.

Main Results:

  • Pol eta competes with other polymerases, reducing overall replication fidelity.
  • Pol eta exhibits low processivity and inefficient extension of mismatched primer termini.
  • Pol eta-induced replication infidelity is modulated by dNTP concentration and enhanced by dGMP, indicating exonucleolytic proofreading.
  • These findings support the role of extrinsic exonucleases in correcting Pol eta errors.

Conclusions:

  • Pol eta's relaxed base selectivity aids UV photoproduct bypass.
  • Subsequent proofreading by extrinsic exonucleases mitigates Pol eta's mutagenic potential.
  • Pol eta's unique properties are optimized for its role in DNA repair and preventing UV-induced skin cancer.

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