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Results for “replication fork remodeling”

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At least 19 recordsLinked to original sources

PubMed2026

Exploiting DNA damage tolerance for precision oncology.

Unresolved DNA lesions trigger replication stress, forcing cancer cells to hijack DNA damage tolerance (DDT) networks, specifically translesion synthesis (TLS) and template switching, to sustain replication. While DDT prevents lethal fork collapse, error-prone TLS drives mutagenesis, tumor evolution, chemoresistance and radioresistance. Proliferating cell nuclear antigen post-translational modifications dynamically govern pathway selection. Cancer cells exploit this plasticity, creating actionable vulnerabilities such as postreplicative single-stranded DNA gaps. Emerging inhibitors targeting TLS polymerases, upstream regulators such as ubiquitin-specific peptidase 1 (USP1), and critical protein-protein interactions offer unprecedented opportunities for precision oncology. By integrating DDT inhibition with biomarkers such as homologous recombination deficiency and tumor mutational burden, we can drive synthetic lethality, sensitize tumors to genotoxic agents, suppress treatment-induced mutagenesis, and potentially enhance responses to immunotherapy.

DDT↗

PubMed2006

Chromatin-remodelling factors and the maintenance of transcriptional states through DNA replication.

At the replication fork, nucleosomes, transcription factors and RNA polymerases are stripped off the DNA, the DNA double strands are unzipped and DNA methylation marks may be erased. Therefore DNA replication is both a 'curse' and 'bliss' for the epigenome, as it disrupts its stability by causing chromatin perturbations, yet it offers an opportunity to initiate changes in chromatin architecture and gene expression patterns, especially during development. Thus the DNA replication site is a critical point for regulation. It has become apparent that there is a close functional relationship between those factors that regulate transcriptional competence and the DNA replication programme. In this review we discuss novel insights into how chromatin-remodelling factors at replication sites are involved in both the maintenance and regulation of transcriptional states.

Binding Sites↗

PubMed2006

Methods to study replication fork collapse in budding yeast.

Replication of the eukaryotic genome is a difficult task, as cells must coordinate chromosome replication with chromatin remodeling, DNA recombination, DNA repair, transcription, cell cycle progression, and sister chromatid cohesion. Yet, DNA replication is a potentially genotoxic process, particularly when replication forks encounter a bulge in the template: forks under these conditions may stall and restart or even break down leading to fork collapse. It is now clear that fork collapse stimulates chromosomal rearrangements and therefore represents a potential source of DNA damage. Hence, the comprehension of the mechanisms that preserve replication fork integrity or that promote fork collapse are extremely relevant for the understanding of the cellular processes controlling genome stability. Here we describe some experimental approaches that can be used to physically visualize the quality of replication forks in the yeast S. cerevisiae and to distinguish between stalled and collapsed forks.

Blotting, Southern↗

PubMed2001

Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae.

We have identified and characterized an alternative RFC complex RFC(Ctf18p, Ctf8p, Dcc1p) that is required for sister chromatid cohesion and faithful chromosome transmission. Ctf18p, Ctf8p, and Dcc1p interact physically in a complex with Rfc2p, Rfc3p, Rfc4p, and Rfc5p but not with Rfc1p or Rad24p. Deletion of CTF18, CTF8, or DCC1 singly or in combination (ctf18Deltactf8Deltadcc1Delta) leads to sensitivity to microtubule depolymerizing drugs and a severe sister chromatid cohesion defect. Furthermore, temperature-sensitive mutations in RFC4 result in precocious sister chromatid separation. Our results highlight a novel function of the RFC proteins and support a model in which sister chromatid cohesion is established at the replication fork via a polymerase switching mechanism and a replication-coupled remodeling of chromatin.

Benomyl↗

PubMed2026

R-loops and D-loops: a delicate balance in genomic stability and instability.

R-loops and D-loops are three-stranded nucleic acid structures that have emerged as central regulators of genome stability, gene expression, and DNA metabolism. R-loops form co-transcriptionally or post-transcriptionally when nascent RNA re-anneals with the template DNA strand, generating an RNA: DNA hybrid that displaces the non-template strand into a single-stranded state. These structures are enriched at CpG island promoters, transcription termination sites, and immunoglobulin class-switch regions, where they coordinate transcription regulation, chromatin remodeling, and DNA damage signaling. D-loops are formed when a single-stranded DNA segment pairs with one strand of a duplex and displaces the other, arising through context-dependent mechanisms that include RAD51- or DMC1-mediated strand invasion in homologous recombination, shelterin-assisted invasion at telomeres, and replication-coupled strand displacement at the mitochondrial DNA origin. They serve as indispensable intermediates in double-strand break repair, telomere maintenance, and mitochondrial DNA replication. Recent cryo-electron microscopy studies have resolved the stepwise RAD51-mediated strand exchange mechanism at near-atomic resolution, substantially advancing structural understanding of D-loop biogenesis. Despite their differences in molecular composition, both structures remodel Watson-Crick base pairing and, when dysregulated, are associated with replication fork stalling, transcription-replication conflicts, and aberrant recombination. This review systematically compares the structural features, formation mechanisms, regulatory networks, and biological functions of R-loops and D-loops, with emphasis on their convergent roles in safeguarding genome integrity. We further discuss rapidly evolving detection technologies and emerging therapeutic strategies targeting these structures in cancer and neurodegeneration, identifying key unresolved questions for future investigation.

Genomic Instability↗

PubMed2006

The HIRAN domain and recruitment of chromatin remodeling and repair activities to damaged DNA.

Aided by sensitive sequence profile searches we identify a novel conserved domain in the N-terminal regions of the SWI2/SNF2 proteins typified by HIP116 and Rad5p (hence HIP116, Rad5p N-terminal domain: HIRAN domain). We show that the HIRAN domain is found as a standalone protein in several bacteria and prophages, or fused to other catalytic domains, such as a nuclease of the restriction endonuclease fold and TDP1-like DNA phosphoesterases, in the eukaryotes. Based on a network of contextual connections in the form of domain architectures, conserved gene neighborhoods and functional interactions we predict that the HIRAN domain is likely to function as a DNA-binding domain that probably recognizes features associated with damaged DNA or stalled replication forks. It might thus act as a sensor to initiate a damaged DNA checkpoint and engage different DNA repair and chromatin remodeling or modifying activities to these sites. In evolutionary terms, the fusion of the HIRAN domain, and the functionally analogous RAD18 Zn-finger and the PARP-type Zn-finger to SWI2/SNF2 ATPases appears to have been a notable factor for recruiting these ATPases for chromatin modification and remodeling in the context of DNA repair.

Amino Acid Sequence↗

PubMed2006

DNA repeat rearrangements mediated by DnaK-dependent replication fork repair.

We propose that rearrangements between short tandem repeated sequences occur by errors made during a replication fork repair pathway involving a replication template switch. We provide evidence here that the DnaK chaperone of E. coli controls this template switch repair process. Mutants in dnaK are sensitive to replication fork damage and exhibit high expression of the SOS response, indicative of repair deficiency. Deletion and expansion of tandem repeats that occur by replication misalignment ("slippage") are also DnaK dependent. Because mutations in dnaX encoding the gamma and tau subunits of DNA polymerase III mimic dnaK phenotypes and are genetically epistatic, we propose that the DnaKJ chaperone remodels the replisome to facilitate repair. The fork remains largely intact because PriA or PriC restart proteins are not required. We also suggest that the poorly defined RAD6-RAD18-RAD5 mechanism of postreplication repair in eukaryotes occurs by an analogous mechanism to the DnaK template-switch pathway in prokaryotes.

DNA Polymerase III↗

PubMed2003

The crystal structure of the bifunctional primase-helicase of bacteriophage T7.

Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA. The lynchpin of the T7 replication system is a bifunctional primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand. We have determined a 3.45 A crystal structure of the T7 primase-helicase that shows an articulated arrangement of the primase and helicase sites. The crystallized primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed primase domains. This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity. The double-jointed structure of the primase-helicase permits a free range of motion for the primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.

Bacteriophage T7↗

PubMed1998

DNA topoisomerase II sites in the histone H4 gene during the highly synchronous cell cycle of Physarum polycephalum.

The nearly perfect synchrony of nuclear division in a plasmodium of Physarum polycephalum provides a powerful system to analyze topoisomerase II cleavage sites in the course of the cell cycle. The histone H4 locus, whose schedule of replication and transcription is precisely known, was chosen for this analysis. Drug-induced topoisomerase II sites are clustered downstream of the histone H4 gene and appear highly dependent on cell cycle stage. They were only detected in mitosis and at the very beginning of S phase, precisely at the time of replication of the histone H4 region. The sites, which were absent in G2 phase, reappeared at the next mitosis. Remarkably, DNase I hypersensitive sites occurred in nearly the same location, but their schedule was totally different: they were absent in mitosis and present in G2. This schedule follows H4 transcription, which peaks in mid-S phase and in the second part of G2 phase and is off during mitosis. These results suggest that topoisomerase II may not be involved in transcription, but plays a role in remodeling chromatin structure, both during chromosome condensation in prophase/metaphase to allow their decatenation and during chromosome decondensation after metaphase to allow replication fork passage throughout the region.

Animals↗

PubMed1984

Remodeling of sperm chromatin following fertilization: nucleosome repeat length and histone variant transitions in the absence of DNA synthesis.

Within the first cell cycle following fertilization the average nucleosomal repeat length of sea urchin male pronuclear chromatin declines by 30-40 base pairs to a value typical of that found in the embryo. This decline occurs after a lag of about 30 min postfertilization, and is accompanied by replication of the male chromatin and accumulation of cleavage-stage (CS) core histone variants. When replication is inhibited by greater than 95% with aphidicolin, the decline in repeat length still occurs, although it is slightly retarded. The decline in repeat length also occurs when protein synthesis is blocked by greater than 98% and DNA synthesis by 60-70% with emetine. The adjustment of nucleosome repeat length therefore can occur in vivo without extensive movement of replication forks across the length of the chromatin, or normal progression of the cell cycle, and appears to require no proteins synthesized postfertilization. Blocking of DNA synthesis or protein synthesis also does not prevent the normal histone variant transitions involved in male pronuclear chromatin remodeling. Although their accumulation is slowed, CS core variants eventually become the predominant male pronuclear histones in their classes when replication is inhibited. Since a shortening of the average nucleosomal repeat length of approximately 10-20% is not sufficient to account for this large acquisition of CS variants, some of the sperm (Sp) core histones are probably displaced from the replication-blocked pronucleus. Therefore, accumulation of CS H2A and CS H2B are temporally correlated with the repeat length transition, whereas replication, normal progression of the cell cycle, and the early histone transitions involving SpH1 and SpH2B are not.

Animals↗

PubMed2004

The chromatin remodeling complex NoRC and TTF-I cooperate in the regulation of the mammalian rRNA genes in vivo.

The transcription termination factor (TTF)-I is a multifunctional nucleolar protein that terminates ribosomal gene transcription, mediates replication fork arrest and regulates RNA polymerase I transcription on chromatin. TTF-I plays a dual role in rDNA regulation, being involved in both activation and silencing of rDNA transcription. The N-terminal part of TTF-I contains a negative regulatory domain (NRD) that inhibits DNA binding. Here we show that interactions between the NRD and the C-terminal part of TTF-I mask the DNA-binding domain of TTF-I. However, interaction with TIP5, a subunit of the nucleolar chromatin remodeling complex, NoRC, recovers DNA-binding activity. We have mapped the protein domains that mediate the interaction between TTF-I and TIP5. The association of TIP5 with the NRD facilitates DNA binding of TTF-I and leads to the recruitment of NoRC to the rDNA promoter. Thus, TTF-I and NoRC act in concert to silence rDNA transcription.

Animals↗

PubMed2005

In and out: histone variant exchange in chromatin.

Alterations in nucleosome structure affect the accessibility of the DNA and can generate specialized domains of chromatin in the genome. Such changes can be introduced by posttranslational modifications of histones, by chromatin remodeling, or by the incorporation of variants of H2A and H3 into nucleosomes. In contrast to the canonical histones, which are deposited behind the replication fork during S phase, histone variants are incorporated in a process that is independent of DNA replication. Recent studies have shown that distinct multiprotein complexes are responsible for the targeted deposition of histone variants at active genes, centromeres and silent loci. The incorporation of histone variants most probably has epigenetic consequences and contributes to architectural changes in chromosomes.

Amino Acid Sequence↗

PubMed2001

Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle.

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACE An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.

Animals↗

PubMed1999

Developmental regulation of replication fork pausing in Xenopus laevis ribosomal RNA genes.

In early Xenopus embryos, replication forks move along the rRNA genes (rDNA) at a uniform rate and terminate at multiple, apparently random sites. In contrast, a polar replication fork barrier (RFB) is found at the 3' end of the rRNA genes in Xenopus cultured cells. We have now analysed the replication intermediates of Xenopus rDNA from a wide range of developmental stages by 2D gel electrophoresis. Surprisingly, up to 15 different replication fork pausing sites (RFPs) simultaneously appear in the rDNA at the midgastrula stage, when rRNA transcription abruptly increases. They disappear during the neurula stage, except for a polar RFP at the 3' end of Xthe transcription unit, which persists to the tadpole stage. The latter RFP is found at the same location as the RFB in cultured cells; however the arrest of replication forks at this RFP is not absolute, since termination occurs at multiple positions throughout the rDNA repeat. The efficiency of fork arrest at this RFP remains constant from midgastrula to early tadpole, and decreases around hatching. The transient appearance of multiple RFPs at midgastrula may reflect some chromatin remodeling associated with developmental activation of rRNA transcription.

Animals↗

PubMed2004

Is chromatin remodeling required to build sister-chromatid cohesion?

Chromosome segregation during mitosis and meiosis depends on the linkage of sister DNA molecules after replication. These links, known as sister-chromatid cohesion, are provided by a multi-subunit complex called cohesin. Recent papers suggest that chromatin-remodeling complexes also have a role in the generation of sister-chromatid cohesion. It remains unclear whether they do so by facilitating the recruitment of cohesin to specific chromosomal sequences or by modifying an event at replication forks giving rise to cohesion between sister DNAs.

Animals↗

PubMed2006

The F-box protein Dia2 overcomes replication impedance to promote genome stability in Saccharomyces cerevisiae.

The maintenance of DNA replication fork stability under conditions of DNA damage and at natural replication pause sites is essential for genome stability. Here, we describe a novel role for the F-box protein Dia2 in promoting genome stability in the budding yeast Saccharomyces cerevisiae. Like most other F-box proteins, Dia2 forms a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin-ligase complex. Systematic analysis of genetic interactions between dia2Delta and approximately 4400 viable gene deletion mutants revealed synthetic lethal/synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin-remodeling pathways. dia2Delta strains exhibit constitutive activation of the checkpoint kinase Rad53 and elevated counts of endogenous DNA repair foci and are unable to overcome MMS-induced replicative stress. Notably, dia2Delta strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extrachromosomal rDNA circle (ERC) formation, consistent with an observed enrichment of Dia2 in the nucleolus. These results suggest that Dia2 is essential for stable passage of replication forks through regions of damaged DNA and natural fragile regions, particularly the replication fork barrier (RFB) of rDNA repeat loci. We propose that the SCFDia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome.

Cell Cycle↗

PubMed2003

Evidence for a role of MCM (mini-chromosome maintenance)5 in transcriptional repression of sub-telomeric and Ty-proximal genes in Saccharomyces cerevisiae.

The MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae. In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains. This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier. We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin. We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor). We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons.

Blotting, Northern↗

PubMed2006

Eukaryotic DNA replication in a chromatin context.

There has been remarkable progress in the last 20 years in defining the molecular mechanisms that regulate initiation of DNA synthesis in eukaryotic cells. Replication origins in the DNA nucleate the ordered assembly of protein factors to form a prereplication complex (preRC) that is poised for DNA synthesis. Transition of the preRC to an active initiation complex is regulated by cyclin-dependent kinases and other signaling molecules, which promote further protein assembly and activate the mini chromosome maintenance helicase. We will review these mechanisms and describe the state of knowledge about the proteins involved. However, we will also consider an additional layer of complexity. The DNA in the cell is packaged with histone proteins into chromatin. Chromatin structure provides an additional layer of heritable information with associated epigenetic modifications. Thus, we will begin by describing chromatin structure, and how the cell generally controls access to the DNA. Access to the DNA requires active chromatin remodeling, specific histone modifications, and regulated histone deposition. Studies in transcription have revealed a variety of mechanisms that regulate DNA access, and some of these are likely to be shared with DNA replication. We will briefly describe heterochromatin as a model for an epigenetically inherited chromatin state. Next, we will describe the mechanisms of replication initiation and how these are affected by constraints of chromatin. Finally, chromatin must be reassembled with appropriate modifications following passage of the replication fork, and our third major topic will be the reassembly of chromatin and its associated epigenetic marks. Thus, in this chapter, we seek to bring together the studies of replication initiation and the studies of chromatin into a single holistic narrative.

Animals↗