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

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE87356 | GEO | 2018/07/27

REPOSITORIES: GEO

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Rtt105 promotes DNA replication by functioning as an RPA chaperone

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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A role of RPA in replication coupled nucleosome assembly

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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

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

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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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PHOSPHORYLATION-DEPENDENT ASSOCIATION OF WRN WITH RPA IS REQUIRED FOR RECOVERY OF REPLICATION FORKS STALLED AT SECONDARY DNA STRUCTURES

Project description:The WRN protein mutated in the premature aging disorder Werner syndrome is vital for metabolism of perturbed replication forks. Replication Protein A (RPA) robustly enhances WRN helicase activity in specific cases when tested in vitro. However, the significance of RPA-binding to WRN in vivo has remained largely unexplored. We identified several conserved phosphorylation sites in the acidic domain of WRN targeted by Casein Kinase 2 (CK2). These sites are crucial for WRN-RPA interaction in vitro and in human cells. Using the CK2-unphosphorylatable WRN mutant lacking the ability to bind RPA, we determined that the WRN-RPA complex plays a critical role in fork recovery after replication stress whereas is not necessary for the processing of replication forks or preventing DNA damage when forks stall or collapse. RPA-binding by WRN and its helicase activity are crucial for countering the persistence of G4 structures after fork stalling. Absence of WRN-RPA binding hampers fork recovery, causing single-strand DNA gaps, enlarged by MRE11, and triggering MUS81-dependent double-strand breaks which requires efficient repair by RAD51 to prevent excessive DNA damage.

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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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2013-01-02 | E-GEOD-42451 | biostudies-arrayexpress

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

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