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Rtt105 functions as a chaperone for Replication Protein A to preserve genome stability

ABSTRACT: Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here we show that the S. cerevisiae protein Regulator of Ty1 Transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal 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 the nucleus and facilitates its loading onto ssDNA at replication forks.

SUBMITTER: Shuqi Li

PROVIDER: S-SCDT-EMBOJ-2018-99154 | biostudies-other |

REPOSITORIES: biostudies-other

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Rtt105 functions as a chaperone for replication protein A to preserve genome stability.

Project description:Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal 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 the nucleus and facilitates its loading onto ssDNA at replication forks.

| S-EPMC6120668 | biostudies-literature

Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability.

Project description:Chromatin structure affects DNA replication patterns, but the role of specific chromatin modifiers in regulating the replication process is yet unclear. We report that phosphorylation of the human SIRT1 deacetylase on Threonine 530 (T530-pSIRT1) modulates DNA synthesis. T530-pSIRT1 associates with replication origins and inhibits replication from a group of 'dormant' potential replication origins, which initiate replication only when cells are subject to replication stress. Although both active and dormant origins bind T530-pSIRT1, active origins are distinguished from dormant origins by their unique association with an open chromatin mark, histone H3 methylated on lysine 4. SIRT1 phosphorylation also facilitates replication fork elongation. SIRT1 T530 phosphorylation is essential to prevent DNA breakage upon replication stress and cells harboring SIRT1 that cannot be phosphorylated exhibit a high prevalence of extrachromosomal elements, hallmarks of perturbed replication. These observations suggest that SIRT1 phosphorylation modulates the distribution of replication initiation events to insure genomic stability.

| S-EPMC5570034 | biostudies-literature

Transcription shapes DNA replication initiation to preserve genome integrity.

Project description:BackgroundEarly DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.ResultsWe develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage.ConclusionRNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

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TFIP11 competes with the Bloom syndrome helicase for binding to stalled replication forks to preserve genome stability

Project description:Replication fork reversal is a key protective mechanism against replication stress in higher eukaryotic cells and occurs via a series of coordinated enzymatic reactions. The Bloom syndrome gene product, BLM, is a member of the highly conserved RecQ helicase family and has been implicated in this process, but its precise regulation and role remain poorly understood. Here, we show that, upon replication stress, the GCFC domain-containing protein TFIP11 competes with the BLM helicase for association with stalled replication forks, thereby facilitates RAD51-mediated stalled fork reversal. Consequently, loss of TFIP11 results in aberrant accumulation of BLM at stalled forks, which in turn compromises RAD51 recruitment, impairs replication stress-induced fork reversal and slowing, hypersensitizes cells to replication stress-inducing agents, and enhances chromosomal instability. These findings reveal a previously unidentified regulatory mechanism that modulates the activities of BLM and RAD51 at stalled forks and thus genome integrity.

2024-05-24 | PXD042222 | Pride

The conserved histone chaperone Spt6 facilitates DNA replication and mediates genome stability [MCM ChIPseq]

Project description:Histone chaperones are an important class of factors that regulate chromatin accessibility for DNA-templated processes. Spt6 is a conserved histone chaperone and key regulator of transcription and chromatin structure. However, its functions outside of these roles have been little explored. In this work, we demonstrate a role for Spt6 in DNA replication and more broadly as a regulator of genome stability. Spt6 binds the replication machinery and mutations or loss of Spt6 impair DNA replication in vivo. Additionally, spt6 mutants are sensitive to DNA replication stress inducing agents, with increased sensitivity when combined with loss of DNA replication associated factors. Furthermore, spt6 mutants have elevated levels of DNA double strand breaks and are hyper-recombinogenic. R-loops do not appear to be elevated in spt6 mutants. In total, we identify Spt6 as a regulator of genome stability, at least in part through a role for Spt6 in DNA replication.

2023-01-03 | GSE221913 | GEO

Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability

Project description:Chromatin structure affects DNA replication patterns, but the role of specific chromatin modifiers in regulating the replication process is yet unclear. We report that phosphorylation of the human SIRT1 deacetylase on Threonine 530 (T530-pSIRT1) modulates DNA synthesis. T530-pSIRT1 associates with replication origins and inhibits replication from a group of ÒdormantÓ potential replication origins, which initiate replication only when cells are subject to replication stress. Although both active and dormant origins bind T530-pSIRT1, active origins are distinguished from dormant origins by their unique association with an open chromatin mark, histone H3 methylated on lysine 4. SIRT1 phosphorylation also facilitates leading and lagging strand coordination. SIRT1 T530 phosphorylation is essential to prevent DNA breakage upon replication stress and cells harboring SIRT1 that cannot be phosphorylated exhibit a high prevalence of extrachromosomal elements, hallmarks of perturbed replication. These observations suggest that SIRT1 phosphorylation modulates the distribution of replication initiation events to insure genomic stability.

2017-06-02 | GSE94403 | GEO

Cohesin modulates DNA replication to preserve genome integrity

Project description:This SuperSeries is composed of the SubSeries listed below.

2022-07-31 | GSE189762 | GEO

The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability.

Project description:Bacteria and yeast possess one RecQ helicase homolog whereas humans contain five RecQ helicases, all of which are important in preserving genome stability. Three of these, BLM, WRN and RECQL4, are mutated in human diseases manifesting in premature aging and cancer. We are interested in determining to which extent these RecQ helicases function cooperatively. Here, we report a novel physical and functional interaction between BLM and RECQL4. Both BLM and RECQL4 interact in vivo and in vitro. We have mapped the BLM interacting site to the N-terminus of RECQL4, comprising amino acids 361-478, and the region of BLM encompassing amino acids 1-902 interacts with RECQL4. RECQL4 specifically stimulates BLM helicase activity on DNA fork substrates in vitro. The in vivo interaction between RECQL4 and BLM is enhanced during the S-phase of the cell cycle, and after treatment with ionizing radiation. The retention of RECQL4 at DNA double-strand breaks is shortened in BLM-deficient cells. Further, depletion of RECQL4 in BLM-deficient cells leads to reduced proliferative capacity and an increased frequency of sister chromatid exchanges. Together, our results suggest that BLM and RECQL4 have coordinated activities that promote genome stability.

| S-EPMC3413146 | biostudies-literature

Abro1 maintains genome stability and limits replication stress by protecting replication fork stability.

Project description:Protection of the stalled replication fork is crucial for responding to replication stress and minimizing its impact on chromosome instability, thus preventing diseases, including cancer. We found a new component, Abro1, in the protection of stalled replication fork integrity. Abro1 deficiency results in increased chromosome instability, and Abro1-null mice are tumor-prone. We show that Abro1 protects stalled replication fork stability by inhibiting DNA2 nuclease/WRN helicase-mediated degradation of stalled forks. Depletion of RAD51 prevents the DNA2/WRN-dependent degradation of stalled forks in Abro1-deficient cells. This mechanism is distinct from the BRCA2-dependent fork protection pathway, in which stable RAD51 filament formation prevents MRE11-dependent degradation of the newly synthesized DNA at stalled forks. Thus, our data reveal a new aspect of regulated protection of stalled replication forks that involves Abro1.

| S-EPMC5588928 | biostudies-literature

Transcription shapes DNA replication initiation to preserve genome integrity

Project description:We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones (ERIZs) in both mouse and human cells. The identified ERIZs fall in open chromatin compartments while are mutually exclusive with transcription elongation and occupy mainly non-transcribed regions adjacent to transcribed regions. Furthermore, we reveal that RNA polymerase II actively redistributes the chromatin-encircled mini-chromosome maintenance (MCM) complex but not the origin-recognition complex (ORC) to actively restrict early DNA replication initiation outside of transcribed regions. The coupling of RNA polymerase II and MCM is further validated by detected MCM accumulation and DNA replication initiation after RNA polymerase II stalling via anchoring nuclease-dead Cas9 at the transcribed genes. Importantly, we also find that the orchestration of DNA replication initiation by transcription can efficiently prevent gross DNA damage.

2021-05-20 | GSE174680 | GEO

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