Proteomics

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

ABSTRACT: Break-induced replication (BIR) mediates single-ended DNA double-strand break (DSB) repair. Upon strand invasion into a homologous repair template, BIR is catalysed by a minimal replisome comprising PCNA, DNA polymerase delta, and the Pif1 helicase. Here, we identify an interaction between Polymerase delta and single-stranded DNA (ssDNA)-binding protein RPA mediated by an RPA-binding module (RBM) within the Polymerase delta 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 the affinity of Polymerase delta for RPA-bound BIR intermediates, thereby boosting BIR efficiency. Modelling indicates that Pol32 phospho-RBM-Rfa1 interactions mirror the binding mode of a previously identified Pif1 RBM, and an RBM we identify in the BIR antagonist Mph1. This implies a key role for RPA in the dynamic orchestration of the enzymes mediating BIR.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Rowin Appanah  

LAB HEAD: Ulrich Rass

PROVIDER: PXD074712 | Pride | 2026-06-15

REPOSITORIES: Pride

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SAMPLE1_02_10_2025_Rim11_2h.msf Msf
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SAMPLE2_02_10_2025_Cdc5_2h.msf Msf
SAMPLE2_02_10_2025_Cdc5_2h.raw Raw
SAMPLE3_02_10_2025_Cka2_2h.msf Msf
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Publications

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

Jones David D   Appanah Rowin R   Yates Luke A LA   Oliver Antony W AW   Rass Ulrich U  

Nature communications 20260409 1

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 phosphoryla  ...[more]

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