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<p>DNA polymerase theta (Pol θ) initiates repair of DNA double-strand breaks by pairing single strands at short “microhomologies”. It is important to understand microhomology selection, as some cancer cells rely on Pol θ for survival. Here, we investigate end-joining by purified human Pol θ, employing DNA sequencing of products generated from oligonucleotide libraries having diverse 3′ ends. Pol θ overwhelmingly selects short internal microhomologies found within 15 nucleotides of the terminus of single-stranded DNAs, restricting deletion size during end-joining. Significantly, we find that the selected microhomologies are usually interrupted by mismatches and that base pairing within 6 nucleotides of the 3′ end is important for determining microhomology choice. Bidirectional synthesis is not necessary to initiate end-joining. The preference for mismatched microhomologies suggests a revision of the definition of microhomology to account for the unique properties of Pol θ. This could advance the analysis of mutations in cancer genomes.</p>

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Pol θ-mediated end-joining uses microhomologies containing mismatches

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月2日

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  • Yuzhen Li,
  • Ngoc K. Dang,
  • Wei He,
  • Mark Returan,
  • Denisse Carvajal-Maldonado,
  • Adele T. Guerin,
  • Han Xu,
  • Bin Liu,
  • Richard D. Wood

摘要

DNA polymerase theta (Pol θ) initiates repair of DNA double-strand breaks by pairing single strands at short “microhomologies”. It is important to understand microhomology selection, as some cancer cells rely on Pol θ for survival. Here, we investigate end-joining by purified human Pol θ, employing DNA sequencing of products generated from oligonucleotide libraries having diverse 3′ ends. Pol θ overwhelmingly selects short internal microhomologies found within 15 nucleotides of the terminus of single-stranded DNAs, restricting deletion size during end-joining. Significantly, we find that the selected microhomologies are usually interrupted by mismatches and that base pairing within 6 nucleotides of the 3′ end is important for determining microhomology choice. Bidirectional synthesis is not necessary to initiate end-joining. The preference for mismatched microhomologies suggests a revision of the definition of microhomology to account for the unique properties of Pol θ. This could advance the analysis of mutations in cancer genomes.