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<p>Break-induced replication (BIR) facilitates single-ended DNA double-strand break (DSB) repair. Upon homologous recombination-mediated strand invasion into a homologous repair template, BIR is catalysed by a minimal replisome comprising PCNA, DNA polymerase δ (Pol δ), and the Pif1 helicase. Here, we identify an interaction between Pol δ and single-stranded DNA (ssDNA)-binding protein RPA mediated by an RPA-binding module (RBM) within Pol δ subunit Pol32 and RPA subunit Rfa1. Pol32 RBM phosphorylation at Thr256 and Thr257 increases its affinity for Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in vivo. This suggests that Pol32 functions as a rheostat whose phosphorylation enhances Pol δ’s affinity for RPA-bound BIR intermediates, thereby boosting BIR efficiency. Modelling indicates that Pol32 phospho-RBM-Rfa1 interactions mirror the binding mode of RBMs in Pif1 and the FANCM helicase and BIR antagonist Mph1. This implies a key role for RPA in the dynamic orchestration of the enzymes mediating BIR.</p>

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Break-induced replication is enhanced by a phospho-activated RPA-binding module in Pol32

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月9日

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  • David Jones,
  • Rowin Appanah,
  • Luke A. Yates,
  • Antony W. Oliver,
  • Ulrich Rass

摘要

Break-induced replication (BIR) facilitates single-ended DNA double-strand break (DSB) repair. Upon homologous recombination-mediated strand invasion into a homologous repair template, BIR is catalysed by a minimal replisome comprising PCNA, DNA polymerase δ (Pol δ), and the Pif1 helicase. Here, we identify an interaction between Pol δ and single-stranded DNA (ssDNA)-binding protein RPA mediated by an RPA-binding module (RBM) within Pol δ subunit Pol32 and RPA subunit Rfa1. Pol32 RBM phosphorylation at Thr256 and Thr257 increases its affinity for Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in vivo. This suggests that Pol32 functions as a rheostat whose phosphorylation enhances Pol δ’s affinity for RPA-bound BIR intermediates, thereby boosting BIR efficiency. Modelling indicates that Pol32 phospho-RBM-Rfa1 interactions mirror the binding mode of RBMs in Pif1 and the FANCM helicase and BIR antagonist Mph1. This implies a key role for RPA in the dynamic orchestration of the enzymes mediating BIR.