Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The functional significance of the RPA- and PCNA-dependent recruitment of Pif1 to DNA

ABSTRACT: Pif1 family helicases are multifunctional proteins conserved in eukaryotes, from yeast to humans. They are important for the genome maintenance in both nuclei and mitochondria, where they have been implicated in Okazaki fragment processing, replication fork progression and termination, telomerase regulation and DNA repair. While the Pif1 helicase activity is readily detectable on naked nucleic acids in vitro, the in vivo functions rely on recruitment to DNA. We identify the single-stranded DNA binding protein complex RPA as the major recruiter of Pif1 in budding yeast, in addition to the previously reported Pif1-PCNA interaction. The two modes of the Pif1 recruitment act independently during telomerase inhibition, as the mutations in the Pif1 motifs disrupting either of the recruitment pathways act additively. In contrast, both recruitment mechanisms are essential for the replication-related roles of Pif1 at conventional forks and during the repair by break-induced replication. We propose a molecular model where RPA and PCNA provide a double anchoring of Pif1 at replication forks, which is essential for the Pif1 functions related to the fork movement.

SUBMITTER: Dr. Svetlana Makovets

PROVIDER: S-SCDT-10_1038-S44319-024-00114-9 | biostudies-other |

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

The functional significance of the RPA- and PCNA-dependent recruitment of Pif1 to DNA.

Project description:Pif1 family helicases are multifunctional proteins conserved in eukaryotes, from yeast to humans. They are important for the genome maintenance in both nuclei and mitochondria, where they have been implicated in Okazaki fragment processing, replication fork progression and termination, telomerase regulation and DNA repair. While the Pif1 helicase activity is readily detectable on naked nucleic acids in vitro, the in vivo functions rely on recruitment to DNA. We identify the single-stranded DNA binding protein complex RPA as the major recruiter of Pif1 in budding yeast, in addition to the previously reported Pif1-PCNA interaction. The two modes of the Pif1 recruitment act independently during telomerase inhibition, as the mutations in the Pif1 motifs disrupting either of the recruitment pathways act additively. In contrast, both recruitment mechanisms are essential for the replication-related roles of Pif1 at conventional forks and during the repair by break-induced replication. We propose a molecular model where RPA and PCNA provide a double anchoring of Pif1 at replication forks, which is essential for the Pif1 functions related to the fork movement.

| S-EPMC11014909 | biostudies-literature

Role of the Pif1-PCNA Complex in Pol ?-Dependent Strand Displacement DNA Synthesis and Break-Induced Replication.

Project description:The S. cerevisiae Pif1 helicase functions with DNA polymerase (Pol) ? in DNA synthesis during break-induced replication (BIR), a conserved pathway responsible for replication fork repair and telomere recombination. Pif1 interacts with the DNA polymerase processivity clamp PCNA, but the functional significance of the Pif1-PCNA complex remains to be elucidated. Here, we solve the crystal structure of PCNA in complex with a non-canonical PCNA-interacting motif in Pif1. The structure guides the construction of a Pif1 mutant that is deficient in PCNA interaction. This mutation impairs the ability of Pif1 to enhance DNA strand displacement synthesis by Pol ? in vitro and also the efficiency of BIR in cells. These results provide insights into the role of the Pif1-PCNA-Pol ? ensemble during DNA break repair by homologous recombination.

| S-EPMC5842794 | biostudies-literature

Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis.

Project description:Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.

| S-EPMC7681027 | biostudies-literature

The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening.

Project description:Pif1 family helicases are evolutionary conserved 5'-3' DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.

| S-EPMC4252562 | biostudies-literature

Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection.

Project description:The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.

| S-EPMC6176631 | biostudies-literature

RPA-mediated recruitment of Bre1 couples histone H2B ubiquitination to DNA replication and repair.

Project description:The ubiquitin E3 ligase Bre1-mediated H2B monoubiquitination (H2Bub) is essential for proper DNA replication and repair in eukaryotes. Deficiency in H2Bub causes genome instability and cancer. How the Bre1-H2Bub pathway is evoked in response to DNA replication or repair remains unknown. Here, we identify that the single-stranded DNA (ssDNA) binding factor RPA acts as a key mediator that couples Bre1-mediated H2Bub to DNA replication and repair in yeast. We found that RPA interacts with Bre1 in vitro and in vivo, and this interaction is stimulated by ssDNA. This association ensures the recruitment of Bre1 to replication forks or DNA breaks but does not affect its E3 ligase activity. Disruption of the interaction abolishes the local enrichment of H2Bub, resulting in impaired DNA replication, response to replication stress, and repair by homologous recombination, accompanied by increased genome instability and DNA damage sensitivity. Notably, we found that RNF20, the human homolog of Bre1, interacts with RPA70 in a conserved mode. Thus, RPA functions as a master regulator for the spatial-temporal control of H2Bub chromatin landscape during DNA replication and recombination, extending the versatile roles of RPA in guarding genome stability.

| S-EPMC7923545 | biostudies-literature

Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis.

Project description:Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.

| S-EPMC3594751 | biostudies-literature

RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand.

Project description:In Saccharomyces cerevisiae, the absence of Pif1 helicase induces the instability of G4-containing CEB1 minisatellite during leading strand but not lagging strand replication. We report that RPA and Pif1 cooperate to maintain CEB1 stability when the G4 forming strand is either on the leading or lagging strand templates. At the leading strand, RPA acts in the same pathway as Pif1 to maintain CEB1 stability. Consistent with this result, RPA co-precipitates with Pif1. This association between Pif1 and RPA is affected by the rfa1-D228Y mutation that lowers the affinity of RPA in particular for G-rich single-stranded DNA. At the lagging strand, in contrast to pif1?, the rfa1-D228Y mutation strongly increases the frequency of CEB1 rearrangements. We explain that Pif1 is dispensable at the lagging strand DNA by the ability of RPA by itself to prevent formation of stable G-rich secondary structures during lagging strand synthesis. Remarkably, overexpression of Pif1 rescues the instability of CEB1 at the lagging strand in the rfa1-D228Y mutant indicating that Pif1 can also act at the lagging strand. We show that the effects of the rfa1-D228Y (rpa1-D223Y in fission yeast) are conserved in Schizosaccharomyces pombe. Finally, we report that RNase H1 interacts in a DNA-dependent manner with RPA in budding yeast, however overexpression of RNase H1 does not rescue CEB1 instability observed in pif1? and rfa1-D228Y mutants. Collectively these results add new insights about the general role of RPA in preventing formation of DNA secondary structures and in coordinating the action of factors aimed at resolving them.

| S-EPMC7063842 | biostudies-literature

RFWD3 and translesion DNA polymerases contribute to PCNA modification-dependent DNA damage tolerance.

Project description:DNA damage tolerance pathways are regulated by proliferating cell nuclear antigen (PCNA) modifications at lysine 164. Translesion DNA synthesis by DNA polymerase η (Polη) is well studied, but less is known about Polη-independent mechanisms. Illudin S and its derivatives induce alkyl DNA adducts, which are repaired by transcription-coupled nucleotide excision repair (TC-NER). We demonstrate that in addition to TC-NER, PCNA modification at K164 plays an essential role in cellular resistance to these compounds by overcoming replication blockages, with no requirement for Polη. Polκ and RING finger and WD repeat domain 3 (RFWD3) contribute to tolerance, and are both dependent on PCNA modifications. Although RFWD3 is a FANC protein, we demonstrate that it plays a role in DNA damage tolerance independent of the FANC pathway. Finally, we demonstrate that RFWD3-mediated cellular survival after UV irradiation is dependent on PCNA modifications but is independent of Polη. Thus, RFWD3 contributes to PCNA modification-dependent DNA damage tolerance in addition to translesion DNA polymerases.

| S-EPMC9348633 | biostudies-literature

Unravelling the clinicopathological and functional significance of replication protein A (RPA) heterotrimeric complex in breast cancers.

Project description:Replication Protein A (RPA), a heterotrimeric complex consisting of RPA1, 2, and 3 subunits, is a single-stranded DNA (ssDNA)-binding protein that is critically involved in replication, checkpoint regulation and DNA repair. Here we have evaluated RPA in 776 pure ductal carcinomas in situ (DCIS), 239 DCIS that co-exist with invasive breast cancer (IBC), 50 normal breast tissue and 4221 IBC. Transcriptomic [METABRIC cohort (n = 1980)] and genomic [TCGA cohort (n = 1090)] evaluations were completed. Preclinically, RPA deficient cells were tested for cisplatin sensitivity and Olaparib induced synthetic lethality. Low RPA linked to aggressive DCIS, aggressive IBC, and shorter survival outcomes. At the transcriptomic level, low RPA tumours overexpress pseudogene/lncRNA as well as genes involved in chemical carcinogenesis, and drug metabolism. Low RPA remains linked with poor outcome. RPA deficient cells are sensitive to cisplatin and Olaparib induced synthetic lethality. We conclude that RPA directed precision oncology strategy is feasible in breast cancers.

| S-EPMC10063624 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data