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Ubiquitin chain editing promotes p97/VCP dependent removal of RPA from DNA

ABSTRACT: How eukaryotic cells choose which DNA repair pathways to use is incompletely understood, yet these decisions are critical to maintain genome stability. Here, we show how error-free DNA repair is controlled by a ubiquitin-dependent mechanism. Central to this is a multi-component ubiquitin chain editing complex, consisting at its core of the single-stranded DNA (ssDNA) binding RPA complex and the ZUP1 deubiquitinase. We show that RPA activates the ZUP1 deubiquitinase towards K63-linkages within diverse ubiquitin chain types and couples this activity to DNA damage recognition. Coupling of these activities is important to control the ubiquitination status of key DNA repair substrates, which allows the RPA-ZUP1 ubiquitin chain editing complex both to balance the usage of error-free DNA repair pathways and prevent toxic protein trapping on DNA. The ubiquitin chain editing activities of the RPA-ZUP1 complex are fine-tuned by ubiquitination of the ZUP1 enzyme, which functions to recruit the p97/VCP ubiquitin segregase and extract it from DNA. We find that loss of the RPA-ZUP1 complex leads to genomic instability in cancer cells and creates vulnerabilities to chemotherapies. Our findings reveal how linkage-specific ubiquitin chain editing can both prevent protein trapping on ssDNA and be used to balance ubiquitin-dependent DNA repair pathway usage. We propose that inhibiting the RPA-ZUP1 complex might be exploited to target diseases with defects in error-free DNA repair pathways or DNA replication stress responses.

ORGANISM(S): Homo sapiens

PROVIDER: GSE289609 | GEO | 2026/09/16

REPOSITORIES: GEO

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Ubiquitin chain editing promotes p97/VCP-dependent removal of RPA from DNA (cross-linking mass spectrometry)

Project description:DNA repair requires dynamic control of proteins on single-stranded DNA (ssDNA), yet how ubiquitin signaling regulates ssDNA-bound factors remains poorly understood. Here we identify in human cells a ubiquitin chain editing mechanism that promotes extraction of replication protein A (RPA) from ssDNA. We show that RPA stimulates the deubiquitinase ZUP1 and enhances its activity toward K63 linkages within ubiquitin chains. In response to DNA damage, RPA is modified with branched K48-K63 ubiquitin chains. ZUP1 selectively removes K63 linkages from these chains, remodeling the ubiquitin signal on RPA. We show that ZUP1-mediated editing of branched ubiquitin chains promotes p97/VCP-dependent removal of ubiquitinated RPA from ssDNA. Loss of ZUP1 causes accumulation of branched ubiquitin chains on RPA and pathological RPA trapping on ssDNA, resulting in elevated ssDNA signaling and genome instability. Together, our findings identify ubiquitin chain editing as a mechanism that controls RPA dynamics on ssDNA and enables p97/VCP-mediated protein extraction during DNA repair

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Ubiquitin chain editing promotes p97/VCP-dependent removal of RPA from DNA

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RNA processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

Project description:Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by HR, suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. Strand-specific transcriptome analysis of biological replicates of WT cells (JKM139 strain) at T0, or 60 and 240 minutes after HO induction, and of xrn1∆, rrp6∆ and trf4∆ cells at T0.

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Project description:Single-stranded DNA (ssDNA) binding protein Replication Protein A (RPA) is essential for protecting ssDNA at replication forks. However, how RPA is loaded to replication forks remains unexplored. Here, we show that Regulator of Ty1 transposition protein 105 (Rtt105) binds RPA and is required for the association of RPA with replication forks. Cells lacking Rtt105 exhibit dramatic genome instability and severe defects in DNA replication. Intriguingly, both the RPA nuclear import and its ability to bind DNA replication forks were greatly compromised in rtt105 mutant cells, however, targeting RPA to the nucleus cannot rescue the defect of RPA binding to replication forks. Importantly, Rtt105 promotes the binding of RPA to ssDNA but does not associate with the final RPA-ssDNA complex in vitro. Moreover, single-molecule studies revealed that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to nucleus and assemble RPA onto ssDNA at replication forks.

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Project description:In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-compartment on damaged chromatin. The molecular organization of this ssDNA- as well as the adjacent dsDNA-compartment is crucial during DSB signaling and repair. However, data regarding the association of the most basic chromatin components – the nucleosomes – have been discrepant. Here, we use site-specific induction of DSBs and chromatin-immunoprecipitation followed by strand-specific sequencing to analyse in vivo binding of key DSB repair and signalling proteins to either the ssDNA- or dsDNA-compartment. In case of nucleosomes, we show that recently proposed ssDNA-nucleosomes are not a major, persistent species, but that nucleosomes eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome- and ssDNA-RPA-compartments during DSB signaling and repair.

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RNA processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

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Ubiquitin chain editing promotes p97/VCP dependent removal of RPA from DNA

Project description:Ubiquitin chain editing promotes p97/VCP dependent removal of RPA from DNA

| PRJNA1223807 | ENA

Replication Protein A1 is essential for DNA damage repair during mammalian oogenesis

Project description:Persistence of unrepaired DNA damage in oocytes is detrimental and may cause genetic aberrations, miscarriage, and infertility. RPA, an ssDNA-binding complex, is essential for various DNA-related processes. Here we report that RPA plays a novel role in DNA damage repair during postnatal oocyte development after meiotic recombination. To investigate the role of RPA during oogenesis, we inactivated RPA1 (replication protein A1), the largest subunit of the heterotrimeric RPA complex, specifically in oocytes using two germline-specific Cre drivers (Ddx4-Cre and Zp3-Cre). We find that depletion of RPA1 leads to the disassembly of the RPA complex, as evidenced by the absence of RPA2 and RPA3 in RPA1-deficient oocytes. Strikingly, severe DNA damage occurs in RPA1-deficient germinal vesicle (GV)-stage oocytes. Loss of RPA in oocytes triggered the canonical DNA damage response mechanisms and pathways, such as activation of ATM, ATR, DNA-PK, and p53. In addition, the RPA deficiency causes chromosome misalignment at metaphase I and metaphase II stages of oocytes, which is consistent with altered transcript levels of genes involved in cytoskeleton organization in RPA1-deficient oocytes. Absence of the RPA complex in oocytes severely impairs folliculogenesis and leads to a significant reduction in oocyte number and female infertility. Our results demonstrate that RPA plays an unexpected role in DNA damage repair during mammalian folliculogenesis.

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