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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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DNA polymerase delta Exo domain stabilizes mononucleotide microsatellites in human cells

Seijiro Shioi1, Akiyoshi Shimamoto1, Yingxia Song2

  • 1Cancer Genetics Laboratory, Clinical Research Institute, National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan.

DNA Repair
|September 16, 2021
PubMed

Insights

DNA polymerase proofreading stabilizes mononucleotide repeats in human cells, unlike dinucleotide repeats. This suggests a division of labor between DNA polymerase and mismatch repair in maintaining genome stability.

Area of Science:

  • Genetics
  • Molecular Biology
  • Genomics

Background:

  • DNA polymerase proofreading (PPR) and DNA mismatch repair (MMR) prevent replication errors in prokaryotes and yeasts.
  • PPR's role in stabilizing repeat sequences in higher eukaryotes is not well-established.
  • Previous studies showed mononucleotide microsatellites destabilized in human cells with mutations in MSH6 and POLD1 Exo domain.

Purpose of the Study:

  • To investigate the role of DNA polymerase delta's Exo domain in stabilizing mononucleotide repeat sequences in human cells.
  • To determine if Exo domain function is essential for mononucleotide repeat stability, independent of MMR.
  • To explore potential functional division between DNA polymerase and MMR in genome maintenance.

Main Methods:

  • Utilized CRISPR/Cas9 gene editing to introduce a specific Exo domain mutation into MSH2-null HeLa cell clones.
  • Compared the stability of mononucleotide and dinucleotide microsatellites in parental MMR-deficient HeLa cells versus genome-edited Exo-/MMR-mutated HeLa clones.
  • Analyzed repeat sequence stability in established cell lines.

Main Results:

  • Mononucleotide repeat sequences were significantly destabilized in Exo-/MMR-mutated HeLa clones, mirroring observations in DLD-1 cells.
  • Dinucleotide microsatellites were destabilized in MMR-deficient backgrounds but showed no significant increase in instability in the genome-edited clones.
  • The Exo domain of DNA polymerase delta plays a crucial role in stabilizing mononucleotide repeats in human cells.

Conclusions:

  • The Exo domain of DNA polymerase delta is essential for maintaining mononucleotide repeat stability in human cells.
  • A functional division likely exists between DNA polymerase and MMR in the maintenance of different types of repeat sequences.
  • These findings provide new insights into the mechanisms of genome stability and repeat maintenance in higher eukaryotes.
Keywords:
DNA mismatch repairMicrosatellitesPolymerase proofreading

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