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Rtt105 promotes DNA replication by functioning as an RPA chaperone

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE101536 | GEO | 2018/07/27

REPOSITORIES: GEO

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Regulator of Ty1 transposition protein 105 (Rtt105) is an RPA chaperone and is required for genome stability maintenance

Project description:Single-stranded DNA (ssDNA) binding protein Replication Protein A (RPA) is essential for protecting ssDNA at replication forks. However, how RPA is guided to DNA replication forks remains unclear. Here, we show that Regulator of Ty1 transposition protein 105 (Rtt105) is an RPA-binding protein and is required for the binding of RPA at replication forks. Cells lacking Rtt105 exhibit extensive genome instability and severe defects in DNA replication. Both the nuclear import of RPA and the extent of genome-wide binding of RPA at replication forks were greatly compromised in rtt105 mutant cells. Mechanistically, Rtt105 is required for the interaction of RPA with Kap95, an importin protein known to be essential for RPA’s nuclear import. Remarkably, Rtt105 strongly enhances RPA binding with ssDNA substrates in vitro. We therefore propose a model in which Rtt105 functions as a RPA chaperone that both escorts RPA during nuclear important and delivers RPA to replication forks.

2018-07-27 | GSE87356 | GEO

A role of RPA in replication coupled nucleosome assembly

Project description:Chromatin replication requires tight coordination of nucleosome assembly machinery with DNA replication machinery. While significant progress has been made in characterizing histone chaperones in this process, the mechanism of whereby nucleosome assembly couples with DNA replication remains largely unknown. Here we show that replication protein A (RPA), a single-stranded DNA (ssDNA) binding protein that is essential for DNA replication provides a binding platform for H3-H4 deposition by histone chaperons and is required for nucleosome formation on nascent chromatin. RPA binds free histone H3-H4 but not nucleosomal histones, and a RPA coated ssDNA stimulates assembly of H3-H4 onto double strand DNA in vitro. RPA mutant with reduced H3-H4 binding exhibits synthetic genetic interaction with mutations at key factors involved in replication-coupled (RC) nucleosome assembly, and are defective in assembly of replicating DNA into nucleosomes in cells. These results reveal a novel function for RPA in nucleosome assembly and a mechanism whereby nucleosome assembly is coordinated with DNA replication.

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The Bloom Syndrome Complex Senses RPA-Coated Single-Stranded DNA to Restart Stalled Replication Forks

Project description:The Bloom syndrome helicase BLM interacts with topoisomerase IIIa (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover products during homologous recombination (HR). BLM also promotes DNA-end resection and stalled replication fork restart. However, it is still unclear how the activities of the BTR complex are regulated in cells. Here, we identify multiple highly conserved short linear peptide motifs within the BTR complex that interact cooperatively with RPA. Furthermore, we demonstrate that RPA-binding is essential for stable recruitment of BLM to DNA damage sites and for stalled fork restart, but not for its roles in HR. Disruption of RPA-binding thus constitutes a novel separation-of-function mutation of the BTR complex, and suggest a model in which BTR contains the intrinsic ability to measure levels of RPA-ssDNA at replication forks, which controls its recruitment and activation in response to replication stress.

2021-01-05 | PXD018322 | Pride

RPA promotes processive ssDNA digestion by Dna2 via their bimodal interaction in Saccharomyces cerevisiae

Project description:Single-stranded DNA (ssDNA) widely exists as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors out-compete RPA and access to ssDNA. Using the budding yeast, Saccharomyces cerevisiae, as a model system, we discovered that Dna2, a key nuclease in DNA replication and repair, employs a bimodal interface to act with RPA both in cis and in trans. The cis-action renders RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA actively delivers its bound ssDNA to Dna2. The trans-action mediated by an acidic patch from Dna2, on the other hand, enables Dna2 to operatie with a sub-optimal amount of RPA or to overcome DNA secondary structures. Genetically, this trans-action mode is not required for cell viability, but indispensable for successful DSB repair.

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Sub1 and RPA associate with RNA Polymerase II at different stages of transcription

Project description:Single stranded DNA binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) pre-initiation complexes (PICs) identified Sub1 and the Replication Protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA binding mutant, suggesting competition for a common binding site. We propose that Sub1 and RPA interact with the non-template strand of RNApII complexes during initiation and elongation, respectively. Chip-chip from wt and sub1D cells with Rfa1 Chromatin immunoprecipitation (ChIP) of Rfa1 in wt and sub1D yeast demonstrated that Rfa1 localization correlates with RNA Polymerase II and is increased at some transcription start sites when Sub1 has been deleted. Comparison of Rfa1 localization in wt vs sub1D yeast

2011-09-26 | E-GEOD-32416 | biostudies-arrayexpress

Sub1 and RPA associate with RNA Polymerase II at different stages of transcription

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Phase separation properties of RPA combine high-affinity ssDNA binding with dynamic condensate functions at telomeres

Project description:RPA has been shown to protect single-stranded DNA (ssDNA) intermediates from instability and breakage. RPA binds ssDNA with sub-nanomolar affinity, yet dynamic turnover is required for downstream ssDNA transactions. How ultrahigh-affinity binding and dynamic turnover are achieved simultaneously is not well understood. Here we reveal that RPA has a strong propensity to assemble into dynamic condensates. In solution, purified RPA phase separates into liquid droplets with fusion and surface wetting behavior. Phase separation is stimulated by sub-stoichiometric amounts of ssDNA, but not RNA or double-stranded DNA, and ssDNA gets selectively enriched in RPA condensates. We find the RPA2 subunit required for condensation and multi-site phosphorylation of the RPA2 N-terminal intrinsically disordered region to regulate RPA self-interaction. Functionally, quantitative proximity proteomics links RPA condensation to telomere clustering and integrity in cancer cells. Collectively, our results suggest that RPA-coated ssDNA is contained in dynamic RPA condensates whose properties are important for genome organization and stability.

2023-03-15 | PXD036935 | Pride

Dna2 removes toxic RPA-ssDNA flaps generated from meiotic recombination-associated DNA synthesis

Project description:During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strand can displace the parental strand to generate single-strand DNA (ssDNA) flaps. Many programmed DSBs and thus many ssDNA flaps occur during meiosis. However, how these flaps are removed remains enigmatic. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) in Saccharomyces cerevisiae causes an abundant accumulation of RPA-ssDNA flaps and an expansion of RPA from DSBs to broader regions. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, inducing Dna2 expression at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreased and increased RPA accumulation in dna2-md, respectively. Together, our findings show that meiotic DSB repair requires Dna2 to remove RPA-ssDNA flaps generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.

2023-08-30 | GSE217628 | GEO

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis

Project description:Repair of DNA double-strand break (DSB) is critical for the maintenance of genome integrity. We have previously shown that a class of DSB-induced small RNAs (diRNAs) facilitates homologous recombination (HR)-mediated DSB repair in Arabidopsis thaliana. Here we show that INVOLVED IN DE NOVO 2 (IDN2), a double-stranded RNA (dsRNA) binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA-binding ARGONAUTE 2 (AGO2) leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from ssDNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair.

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RPA accumulation during class switch recombination represents 5'-3' DNA end resection during the S-G2/M phase of the cell cycle

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