Search Results (16)

R-loops can act as replication fork barriers, creating transcription–replication collisions and inducing replication stress by arresting DNA synthesis, thereby possibly causing aberrant processing and the formation of DNA strand breaks. RNase H1 (RH1) is one of the enzymes that participates in R-loop degradation by cleaving the RNA strand within a hybrid RNA–DNA duplex. In this study, the kinetic features of the interaction of RH1 from Escherichia coli with R-loops of various structures were investigated. It was found that the values of the dissociation constants K_d were minimal for complexes of RH1 with model R-loops containing a 10–11-nt RNA–DNA hybrid part, indicating effective binding. Analysis of the kinetics of RNA degradation in the R-loops by RH1 revealed that the rate-limiting step of the process was catalytic-complex formation. In the presence of RNA polymerase, the R-loops containing a ≤16-nt RNA–DNA hybrid part were efficiently protected from cleavage by RH1. In contrast, R-loops containing longer RNA–DNA hybrid parts, as a model of an abnormal transcription process, were not protected by RNA polymerase and were effectively digested by RH1. Full article

Activation of oncogenes disturbs a wide variety of cellular processes and induces physiological dysregulation of DNA replication, widely referred to as replication stress (RS). Oncogene-induced RS can cause replication forks to stall or collapse, thereby leading to DNA damage. While the DNA damage response (DDR) can provoke an anti-tumor barrier to prevent the development of cancer, a small subset of cells triggers replication stress tolerance (RST), allowing precancerous cells to survive, thereby promoting clonal expansion and genomic instability (GIN). Genomic instability (GIN) is a hallmark of cancer, driving genetic alterations ranging from nucleotide changes to aneuploidy. These alterations increase the probability of oncogenic events and create a heterogeneous cell population with an enhanced ability to evolve. This review explores how major oncogenes such as RAS, cyclin E, and MYC induce RS through diverse mechanisms. Additionally, we delve into the strategies employed by normal and cancer cells to tolerate RS and promote GIN. Understanding the intricate relationship between oncogene activation, RS, and GIN is crucial to better understand how cancer cells emerge and to develop potential cancer therapies that target these vulnerabilities. Full article

BRCA1 is a tumor suppressor with a complex mode of action. Hereditary mutations in BRCA1 predispose carriers to breast cancer, and spontaneous breast cancers often exhibit defects in BRCA1 expression. However, haploinsufficiency or suppression of BRCA1 expression leads to defects in DNA repair, which can induce DNA damage responses, leading to senescence. Activating mutation or overexpression of the Her2 oncoprotein are also frequent drivers of breast cancer. Yet, over-activation of Her2, working through the RAS oncoprotein, can also induce senescence. It is thought that additional defects in the p53 and Rb tumor suppressor machinery must occur in such tumors to allow an escape from senescence, thus permitting tumor development. Although BRCA1 mutant breast cancers are usually Her2 negative, a significant percentage of Her2 positive tumors also lose their expression of BRCA1. Such Her2+/BRCA1− tumors might be expected to have a particularly high senescence barrier to overcome. An important RAS senescence effector is the protein NORE1A, which can modulate both p53 and Rb. It is an essential senescence effector of the RAS oncoprotein, and it is often downregulated in breast tumors by promotor methylation. Here we show that NORE1A forms a Her2/RAS regulated, endogenous complex with BRCA1 at sites of replication fork arrest. Suppression of NORE1A blocks senescence induction caused by BRCA1 inactivation and Her2 activation. Thus, NORE1A forms a tumor suppressor complex with BRCA1. Its frequent epigenetic inactivation may facilitate the transformation of Her2+/BRCA1− mediated breast cancer by suppressing senescence. Full article

Origins of replication are genomic regions in which replication initiates in a bidirectional manner. Recently, a new methodology (origin-derived single-stranded DNA sequencing; ori-SSDS) was developed that allows the detection of replication initiation in a strand-specific manner. Reanalysis of the strand-specific data revealed that 18–33% of the peaks are non-symmetrical, suggesting a single direction of replication. Analysis of replication fork direction data revealed that these are origins of replication in which the replication is paused in one of the directions, probably due to the existence of a replication fork barrier. Analysis of the unidirectional origins revealed a preference of G4 quadruplexes for the blocked leading strand. Taken together, our analysis identified hundreds of genomic locations in which the replication initiates only in one direction, and suggests that G4 quadruplexes may serve as replication fork barriers in such places. Full article

The proper maintenance of genetic material is essential for the survival of living organisms. One of the main safeguards of genome stability is homologous recombination involved in the faithful repair of DNA double-strand breaks, the restoration of collapsed replication forks, and the bypass of replication barriers. Homologous recombination relies on the formation of Rad51 nucleoprotein filaments which are responsible for the homology-based interactions between DNA strands. Here, we demonstrate that without the regulation of these filaments by Srs2 and Rad54, which are known to remove Rad51 from single-stranded and double-stranded DNA, respectively, the filaments strongly inhibit damage-associated DNA synthesis during DNA repair. Furthermore, this regulation is essential for cell survival under normal growth conditions, as in the srs2Δ rad54Δ mutants, unregulated Rad51 nucleoprotein filaments cause activation of the DNA damage checkpoint, formation of mitotic bridges, and loss of genetic material. These genome instability features may stem from the problems at stalled replication forks as the lack of Srs2 and Rad54 in the presence of Rad51 nucleoprotein filaments impedes cell recovery from replication stress. This study demonstrates that the timely and efficient disassembly of recombination machinery is essential for genome maintenance and cell survival. Full article

Pif1 helicases are a multifunctional family of DNA helicases that are important for many aspects of genomic stability in the nucleus and mitochondria. Pif1 helicases are conserved from bacteria to humans. Pif1 helicases play multiple roles at the replication fork, including promoting replication through many barriers such as G-quadruplex DNA, the rDNA replication fork barrier, tRNA genes, and R-loops. Pif1 helicases also regulate telomerase and promote replication termination, Okazaki fragment maturation, and break-induced replication. This review highlights many of the roles and regulations of Pif1 at the replication fork that promote cellular health and viability. Full article

Termination of DNA replication, the final stage of genome duplication, is surprisingly complex, and failures to bring DNA synthesis to an accurate conclusion can impact genome stability and cell viability. In Escherichia coli, termination takes place in a specialised termination area opposite the origin. A ‘replication fork trap’ is formed by unidirectional fork barriers via the binding of Tus protein to genomic ter sites. Such a fork trap system is found in some bacterial species, but it appears not to be a general feature of bacterial chromosomes. The biochemical properties of fork trap systems have been extensively characterised, but little is known about their precise physiological roles. In this study, we compare locations and distributions of ter terminator sites in E. coli genomes across all phylogenetic groups, including Shigella. Our analysis shows that all ter sites are highly conserved in E. coli, with slightly more variability in the Shigella genomes. Our sequence analysis of ter sites and Tus proteins shows that the fork trap is likely to be active in all strains investigated. In addition, our analysis shows that the dif chromosome dimer resolution site is consistently located between the innermost ter sites, even if rearrangements have changed the location of the innermost termination area. Our data further support the idea that the replication fork trap has an important physiological role that provides an evolutionary advantage. Full article

Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation. Full article

Chromosomal instability (CIN) is associated with many human diseases, including neurodevelopmental or neurodegenerative conditions, age-related disorders and cancer, and is a key driver for disease initiation and progression. A major source of structural chromosome instability (s-CIN) leading to structural chromosome aberrations is “replication stress”, a condition in which stalled or slowly progressing replication forks interfere with timely and error-free completion of the S phase. On the other hand, mitotic errors that result in chromosome mis-segregation are the cause of numerical chromosome instability (n-CIN) and aneuploidy. In this review, we will discuss recent evidence showing that these two forms of chromosomal instability can be mechanistically interlinked. We first summarize how replication stress causes structural and numerical CIN, focusing on mechanisms such as mitotic rescue of replication stress (MRRS) and centriole disengagement, which prevent or contribute to specific types of structural chromosome aberrations and segregation errors. We describe the main outcomes of segregation errors and how micronucleation and aneuploidy can be the key stimuli promoting inflammation, senescence, or chromothripsis. At the end, we discuss how CIN can reduce cellular fitness and may behave as an anticancer barrier in noncancerous cells or precancerous lesions, whereas it fuels genomic instability in the context of cancer, and how our current knowledge may be exploited for developing cancer therapies. Full article

Components of the nuclear pore complex (NPC) have been shown to play a crucial role in protecting against replication stress, and recovery from some types of stalled or collapsed replication forks requires movement of the DNA to the NPC in order to maintain genome stability. The role that nuclear positioning has on DNA repair has been investigated in several systems that inhibit normal replication. These include structure forming sequences (expanded CAG repeats), protein mediated stalls (replication fork barriers (RFBs)), stalls within the telomere sequence, and the use of drugs known to stall or collapse replication forks (HU + MMS or aphidicolin). Recently, the mechanism of relocation for collapsed replication forks to the NPC has been elucidated. Here, we will review the types of replication stress that relocate to the NPC, the current models for the mechanism of relocation, and the currently known protective effects of this movement. Full article

DNA interstrand cross-links (ICLs) represent a major barrier blocking DNA replication fork progression. ICL accumulation results in growth arrest and cell death—particularly in cell populations undergoing high replicative activity, such as cancer and leukemic cells. For this reason, agents able to induce DNA ICLs are widely used as chemotherapeutic drugs. However, ICLs are also generated in cells as byproducts of normal metabolic activities. Therefore, every cell must be capable of rescuing lCL-stalled replication forks while maintaining the genetic stability of the daughter cells in order to survive, replicate DNA and segregate chromosomes at mitosis. Inactivation of the Fanconi anemia/breast cancer-associated (FANC/BRCA) pathway by inherited mutations leads to Fanconi anemia (FA), a rare developmental, cancer-predisposing and chromosome-fragility syndrome. FANC/BRCA is the key hub for a complex and wide network of proteins that—upon rescuing ICL-stalled DNA replication forks—allows cell survival. Understanding how cells cope with ICLs is mandatory to ameliorate ICL-based anticancer therapies and provide the molecular basis to prevent or bypass cancer drug resistance. Here, we review our state-of-the-art understanding of the mechanisms involved in ICL resolution during DNA synthesis, with a major focus on how the FANC/BRCA pathway ensures DNA strand opening and prevents genomic instability. Full article

The two PIF1 family helicases in Saccharomyces cerevisiae, Rrm3, and ScPif1, associate with thousands of sites throughout the genome where they perform overlapping and distinct roles in telomere length maintenance, replication through non-histone proteins and G4 structures, lagging strand replication, replication fork convergence, the repair of DNA double-strand break ends, and transposable element mobility. ScPif1 and its fission yeast homolog Pfh1 also localize to mitochondria where they protect mitochondrial genome integrity. In addition to yeast serving as a model system for the rapid functional evaluation of human Pif1 variants, yeast cells lacking Rrm3 have proven useful for elucidating the cellular response to replication fork pausing at endogenous sites. Here, we review the increasingly important cellular functions of the yeast PIF1 helicases in maintaining genome integrity, and highlight recent advances in our understanding of their roles in facilitating fork progression through replisome barriers, their functional interactions with DNA repair, and replication stress response pathways. Full article

Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. Full article

To complete the duplication of large genomes efficiently, mechanisms have evolved that coordinate DNA unwinding with DNA synthesis and provide quality control measures prior to cell division. Minichromosome maintenance protein 10 (Mcm10) is a conserved component of the eukaryotic replisome that contributes to this process in multiple ways. Mcm10 promotes the initiation of DNA replication through direct interactions with the cell division cycle 45 (Cdc45)-minichromosome maintenance complex proteins 2-7 (Mcm2-7)-go-ichi-ni-san GINS complex proteins, as well as single- and double-stranded DNA. After origin firing, Mcm10 controls replication fork stability to support elongation, primarily facilitating Okazaki fragment synthesis through recruitment of DNA polymerase-α and proliferating cell nuclear antigen. Based on its multivalent properties, Mcm10 serves as an essential scaffold to promote DNA replication and guard against replication stress. Under pathological conditions, Mcm10 is often dysregulated. Genetic amplification and/or overexpression of MCM10 are common in cancer, and can serve as a strong prognostic marker of poor survival. These findings are compatible with a heightened requirement for Mcm10 in transformed cells to overcome limitations for DNA replication dictated by altered cell cycle control. In this review, we highlight advances in our understanding of when, where and how Mcm10 functions within the replisome to protect against barriers that cause incomplete replication. Full article

Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair. Full article