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PARP1 auto-modification promotes faithful Okazaki fragment processing and limits replication fork speed

ABSTRACT: Poly(ADP-ribose) polymerase (PARP) inhibitors have proven their efficacy for treating tumors defective in homologous recombination via synthetic lethality. In response to DNA breaks, PARP1 is the primary ADP-ribosylation writer, modifying itself (auto-modification) and other proteins to facilitate repair. However, enzymatic inhibition blocks both processes, making it difficult to dissect their distinct functional roles. Using proteomics and site-directed mutagenesis, we identified a PARP1 mutant deficient in auto-modification, yet it retains catalytic activity. This separation-of-function mutant revealed that PARP1 auto-modification slows DNA replication fork progression but is dispensable for repair factor recruitment. Instead, auto-modification promotes the timely release of PARP1 at DNA break sites and prevents the formation of replication stress. Simultaneous inhibition of FEN1 and loss of PARP1 auto-modification gives rise to synthetic lethality, implicating auto-modification in Okazaki fragment processing. Our results demonstrate that trapping of PARP at DNA breaks impedes repair factor accessibility, constituting an important dimension of PARP-inhibitor-driven cytotoxicity.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jonas Elsborg

LAB HEAD: Michael L.

PROVIDER: PXD066033 | Pride | 2025-10-02

REPOSITORIES: Pride

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			Action	DRS
	20250220_LUMOS-CCG_LC12_JE_lowpH_4SA_ctrl_01.raw	Raw
	20250220_LUMOS-CCG_LC12_JE_lowpH_4SA_ctrl_02.raw	Raw
	20250220_LUMOS-CCG_LC12_JE_lowpH_4SA_ctrl_03.raw	Raw
	20250220_LUMOS-CCG_LC12_JE_lowpH_4SA_ctrl_04.raw	Raw
	20250220_LUMOS-CCG_LC12_JE_lowpH_HPF1ko_ctrl_01.raw	Raw

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PARP1 auto-modification promotes faithful Okazaki fragment processing and limits replication fork speed

Project description:Poly(ADP-ribose) polymerase (PARP) inhibitors have proven their efficacy for treating tumors defective in homologous recombination via synthetic lethality. In response to DNA breaks, PARP1 is the primary ADP-ribosylation writer, modifying itself (auto-modification) and other proteins to facilitate repair. However, enzymatic inhibition blocks both processes, making it difficult to dissect their distinct functional roles. Using proteomics and site-directed mutagenesis, we identified a PARP1 mutant deficient in auto-modification, yet it retains catalytic activity. This separation-of-function mutant revealed that PARP1 auto-modification slows DNA replication fork progression but is dispensable for repair factor recruitment. Instead, auto-modification promotes the timely release of PARP1 at DNA break sites and prevents the formation of replication stress. Simultaneous inhibition of FEN1 and loss of PARP1 auto-modification gives rise to synthetic lethality, implicating auto-modification in Okazaki fragment processing. Our results demonstrate that trapping of PARP at DNA breaks impedes repair factor accessibility, constituting an important dimension of PARP-inhibitor-driven cytotoxicity.

2025-10-02 | PXD052191 | Pride

Preservation of Ester-linked Modifications Reveals Glutamate/Aspartate mono-ADP-ribosylation by PARP1 and its Reversal by PARG

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