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Single-stranded DNA binding proteins influence APOBEC3A substrate preference.

Amber L Brown1, Christopher D Collins1, Secily Thompson1

  • 1School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, WA, USA.

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|October 26, 2021
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Summary
This summary is machine-generated.

The cytidine deaminase, APOBEC3A (A3A), shows higher activity on short hairpin DNA loops. Other proteins and RNA polymerase limit A3A activity on longer single-stranded DNA, explaining mutation patterns in cancer genomes.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • APOBEC3A (A3A) is a key source of cancer mutations, with signatures unevenly distributed in genomes.
  • A3A mutations associate with single-stranded DNA (ssDNA) during replication and hairpin structures, but factors influencing this are unknown.

Purpose of the Study:

  • To investigate the biochemical and cellular factors governing APOBEC3A's specificity for DNA substrates.
  • To elucidate the mechanisms behind the non-uniform distribution of A3A-induced mutations in cancer genomes.

Main Methods:

  • In vitro measurement of A3A cytidine deaminase activity on various ssDNA substrates, including hairpin loops, bubble structures, and replication fork mimics.
  • Assays incorporating whole cell lysates or purified RPA to assess A3A activity in a cellular context.
  • Analysis of A3A activity on ssDNA associated with stalled T7 RNA polymerase.

Main Results:

  • A3A exhibits highest activity on hairpins with 4 nt ssDNA loops, showing only a 2- to fivefold preference over linear ssDNA.
  • Cellular factors like RPA and whole cell lysates significantly reduce A3A activity on linear ssDNA more than on hairpin substrates.
  • A3A activity is diminished at ssDNA bound by stalled T7 RNA polymerase.

Conclusions:

  • The enrichment of A3A mutations in hairpin sequences is partly due to other proteins binding longer ssDNA, limiting A3A access.
  • Protein occlusion, such as by RNA polymerase, contributes to the observed transcriptional strand bias and targeting of hairpin sequences in cancer.
  • These findings provide insights into the mutational landscape of cancer genomes driven by APOBEC3A.