Project description:In this study, we map sites of replication initiation and breakage in primary cells at high resolution under conditions of replication stress. We show that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, large (>20 bp) homopolymeric (dA/dT) tracts are also preferential sites of polar replication fork stalling and collapse. We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes serves to promote replication initiation, but at the cost of increasing chromosome fragility.
Project description:Expression profiles indicate that C-terminal of rep2 is essential for its transactivation activity. Keywords: rep2 mutants cells treated with 8 mM HU for different time points vs wildtype untreated cells We analyzed 40 arrays for rep2 mutants cells treated with 8 mM HU to wild type cells.
Project description:Expression profiles indicate that C-terminal of rep2 is essential for its transactivation activity. Keywords: rep2 mutants cells treated with 8 mM HU for different time points vs wildtype untreated cells
Project description:Hydroxyurea (HU) is toxic to Sulfolobus cells. To address the basis of the HU toxicity, we performed transcriptome analyses on untreated cells and cells following exposure to 5 mM HU for 4 hours.
Project description:To characterize the binding sites of RBMX and RPA on chromatin, 293T cells were treated with CPT and binding profiles were obtained by ChIP-seq assay. RBMX ChIP-seq profile was highly correlated with RPA ChIP-seq profile, both showing strong peaks at centromeres. To characterize the occupancy sites bound by both RBMX and RPA, we annotated their shared peaks. The results showed that these sequences are mostly enriched on repetitive DNAs including rRNA, simple repeats and satellite DNAs.
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.
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