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Results for “Genome Maintenance”

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At least 19 recordsLinked to original sources
PubMed2005

Aging and genome maintenance.

Genomic instability in somatic cells has been implicated as a major stochastic mechanism of aging. Using a transgenic mouse model with chromosomally integrated lacZ mutational target genes, we found mutations to accumulate with age at an organ- and tissue-specific rate. Also the spectrum of age-accumulated mutations was found to differ greatly from organ to organ; while initially similar, mutation spectra of different tissues diverged significantly over the lifetime. To explain how genomic instability, which is inherently stochastic, can be a causal factor in aging, it is proposed that randomly induced mutations may adversely affect normal patterns of gene regulation, resulting in a mosaic of cells at various stages on a trajectory of degeneration, eventually resulting in cell death or neoplastic transformation. To directly address this question we demonstrate that it is now possible to analyze single cells, isolated from old and young tissues, for specific alterations in gene expression.

Aging↗
PubMed2002

Arabidopsis thaliana, a versatile model system for study of eukaryotic genome-maintenance functions.

The genome of the model plant Arabidopsis thaliana encodes many orthologs of human genome-maintenance proteins, and in several important cases plant DNA repair and mutation-antagonism functions resemble their mammalian counterparts more closely than do those of established microbial models. These orthologs, in conjunction with the powerful tools now available for work with Arabidopsis and the practical advantages of its small size and rapid life cycle, now make it an attractive model system for study of eukaryotic DNA repair and mutagenesis. Already, null mutations that inactivate proteins involved in repair of DNA double-strand breaks or in DNA translesion synthesis and are lethal in mice have proved to be tolerated by plants. This review compares in some detail the genome-maintenance activities encoded by plants, mammals and microbes, and describes important Arabidopsis tools and life cycle characteristics. It concludes with selected examples that illustrate Arabidopsis advantages and/or reveal new insights into genome-maintenance functions of general interest.

Animals↗
PubMed2003

Aging and genome maintenance: lessons from the mouse?

Recent progress in the science of aging is driven largely by the use of model systems, ranging from yeast and nematodes to mice. These models have revealed conservation in genetic pathways that balance energy production and its damaging by-products with pathways that preserve somatic maintenance. Maintaining genome integrity has emerged as a major factor in longevity and cell viability. Here we discuss the use of mouse models with defects in genome maintenance for understanding the molecular basis of aging in humans.

Aging↗
PubMed2004

Biased distribution of DNA uptake sequences towards genome maintenance genes.

Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within coding regions are the DNA uptake sequences (DUS) required for natural genetic transformation. More importantly, we found a significantly higher density of DUS within genes involved in DNA repair, recombination, restriction-modification and replication than in any other annotated gene group in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H.influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions. These results imply that the high frequency of DUS in genome maintenance genes is conserved among phylogenetically divergent species and thus are of significant biological importance. Increased DUS density is expected to enhance DNA uptake and the over-representation of DUS in genome maintenance genes might reflect facilitated recovery of genome preserving functions. For example, transient and beneficial increase in genome instability can be allowed during pathogenesis simply through loss of antimutator genes, since these DUS-containing sequences will be preferentially recovered. Furthermore, uptake of such genes could provide a mechanism for facilitated recovery from DNA damage after genotoxic stress.

Bacterial Proteins↗
PubMed2003

Different genome maintenance strategies in human and tobacco cells.

In this work, genome maintenance strategies of organisms belonging to different kingdoms (animals versus plants) but of similar genome size were investigated using a novel, universal double-strand break (DSB) repair assay. Different plasmids linearised with KpnI, Acc65I or EcoRV yielding either 3' or 5' protruding or blunt DNA termini, respectively, were transfected into HeLa cells and Nicotiana plumbaginifolia protoplasts and assayed for the efficiency and fidelity of DSB repair. We show that the mechanism of break sealing is similar but that drastic differences are seen in the fidelity of repair: in HeLa cells, 50-55% DSBs were repaired precisely, compared to as little as 15-30% in tobacco cells. Moreover, the DSB repair in plants resulted in 30-40% longer deletions and significantly shorter insertions. Combined, these led to more than twofold larger net DNA loss in tobacco cells. Our observations point to possible differences in the strategies of DSB repair and genome maintenance in plants and animals.

Cell Line↗
PubMed2001

Multiple regulators of Ty1 transposition in Saccharomyces cerevisiae have conserved roles in genome maintenance.

Most Ty1 retrotransposons in the genome of Saccharomyces cerevisiae are transpositionally competent but rarely transpose. We screened yeast mutagenized by insertion of the mTn3-lacZ/LEU2 transposon for mutations that result in elevated Ty1 cDNA-mediated mobility, which occurs by cDNA integration or recombination. Here, we describe the characterization of mTn3 insertions in 21 RTT (regulation of Ty1 transposition) genes that result in 5- to 111-fold increases in Ty1 mobility. These 21 RTT genes are EST2, RRM3, NUT2, RAD57, RRD2, RAD50, SGS1, TEL1, SAE2, MED1, MRE11, SCH9, KAP122, and 8 previously uncharacterized genes. Disruption of RTT genes did not significantly increase Ty1 RNA levels but did enhance Ty1 cDNA levels, suggesting that most RTT gene products act at a step after mRNA accumulation but before cDNA integration. The rtt mutations had widely varying effects on integration of Ty1 at preferred target sites. Mutations in RTT101 and NUT2 dramatically stimulated Ty1 integration upstream of tRNA genes. In contrast, a mutation in RRM3 increased Ty1 mobility >100-fold without increasing integration upstream of tRNA genes. The regulation of Ty1 transposition by components of fundamental pathways required for genome maintenance suggests that Ty1 and yeast have coevolved to link transpositional dormancy to the integrity of the genome.

Blotting, Northern↗
PubMed2026

The function of Mgs1/WRNIP1 in genome maintenance.

Preservation of genomic integrity during replication is challenging, because replication forks are often stalled by several forms of DNA damage or stable secondary DNA structures. Prolonged stalling of the replication fork can lead to incomplete replication, which may induce double strand breaks, genome rearrangements and cell death. Therefore, several DNA repair mechanisms evolved to rescue stalled replication forks, which can elaborate in error-free or error-prone manners. The pathway selection and the fine tuning of the collaboration between different DNA repair proteins involved in the rescue of the stalled replication fork are very important. Based on our recent knowledge, yeast Mgs1 and its human homologue WRNIP1 proteins can be excellent candidates for this fine-tuning regulator function. In this review we summarize our current knowledge about them and try to point out the most important future steps to prove this hypothesis.

DNA repair↗
PubMed2026

Distinct functions of mammalian RAD51 paralogs in genome maintenance.

RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are evolutionarily conserved essential proteins for cell survival and genome maintenance. RAD51 paralogs were originally identified to play a role in homologous recombination-mediated repair of DNA double-strand breaks (DSBs). However, investigations over the last decade have uncovered new roles of RAD51 paralogs beyond DSB repair in replication stress responses, including replication fork progression, fork stability, and its restart. Recent structural studies have not only uncovered the molecular architecture of previously known RAD51 paralog complexes but also identified novel paralog complex assemblies, providing mechanistic insights into their various genome-maintenance functions. Additionally, a role for RAD51 paralogs in resolving R-loops has been identified, and studies with cancer-associated variants suggest that RAD51 paralogs are potential determinants of cancer susceptibility and therapeutic responses. In the present review, we highlight the recently deciphered structures and novel functions of RAD51 paralog complexes and discuss the clinical and therapeutic implications.

Rad51 Recombinase↗
PubMed2007

Genome maintenance defects in cultured cells and mice following partial inactivation of the essential cell cycle checkpoint gene Hus1.

Cell cycle checkpoints are evolutionarily conserved signaling pathways that uphold genomic integrity. Complete inactivation of the mouse checkpoint gene Hus1 results in chromosomal instability, genotoxin hypersensitivity, and embryonic lethality. To determine the functional consequences of partial Hus1 impairment, we generated an allelic series in which Hus1 expression was incrementally reduced by combining a hypomorphic Hus1 allele, Hus1(neo), with either wild-type or null (Hus1(Delta1)) alleles. Primary Hus1(neo/Delta1) embryonic fibroblasts exhibited spontaneous chromosomal abnormalities and underwent premature senescence, while higher Hus1 expression in Hus1(neo/neo) cells allowed for normal proliferation. Antioxidant treatment almost fully suppressed premature senescence in Hus1(neo/Delta1) cultures, suggesting a critical role for Hus1 in oxidative stress responses. Treatment of Hus1(neo/neo) and Hus1(neo/Delta1) cells with the DNA adducting agent benzo(a)pyrene dihydrodriol epoxide resulted in a loss of cell viability that was associated with S-phase DNA damage checkpoint failure. Likewise, the DNA polymerase inhibitor aphidicolin triggered increased cell death, chromosomal aberrations, and H2AX phosphorylation, a marker for double-stranded DNA breaks, in Hus1(neo/neo) and Hus1(neo/Delta1) cultures compared to controls. Despite these pronounced genome maintenance defects in cultured Hus1(neo/Delta1) and Hus1(neo/neo) cells, mice of the same genotypes were born at expected frequencies and appeared grossly normal. A significant increase in micronucleus formation was observed in peripheral blood cells from Hus1(neo/Delta1) mice, but reduced Hus1 expression did not cause an elevated predisposition to spontaneous tumor development or accelerate tumorigenesis in p53-deficient mice. These results identify differential effects of altered Hus1 gene dosage on genome maintenance during in vitro culture, genotoxic stress responses, embryonic development, and adult homeostasis.

Alleles↗
PubMed2026

Folding a broken genome: the versatile roles of cohesin in genome maintenance.

Cohesin is a protein complex that shapes 3D genome organization through two distinct mechanisms. First, cohesin tethers replicated chromatids from DNA replication until mitosis. This process, known as sister chromatid cohesion, ensures accurate chromosome segregation and enables high-fidelity DNA repair through homologous recombination between the sister chromatids. Second, cohesin organizes the genome during interphase by dynamically extruding chromatin loops, structures that have key roles in gene regulation. Recent work has shown that, in addition to the well-established repair functions of sister chromatid cohesion, cohesin-mediated chromatin looping is closely linked to the repair of DNA double-strand breaks - one of the most toxic DNA lesions. In this Review, we discuss the central roles of cohesin in maintaining genome stability, with emphasis on the cellular response to DNA double-strand breaks. We review how dynamic loop structures facilitate signalling of repair events and promote long-range chromatin motions that underpin the repair process. Overall, its dual mode of action - cohesion and loop extrusion - positions cohesin as a central regulator of chromatin architecture and genome maintenance.

Cohesins↗
PubMed2006

ChlR1 is required for loading papillomavirus E2 onto mitotic chromosomes and viral genome maintenance.

Autonomously replicating DNA viruses must evade mitotic checkpoints and actively partition their genomes to maintain persistent infection. The E2 protein serves these functions by tethering papillomavirus episomes to mitotic chromosomes; however, the mechanism remains unresolved. We show that E2 binds ChlR1, a DNA helicase that plays a role in sister chromatid cohesion. The E2 mutation W130R fails to bind ChlR1 and correspondingly does not associate with mitotic chromosomes. Viral genomes encoding this E2 mutation are not episomally maintained in cell culture. Notably, E2 W130R binds Brd4, which reportedly acts as a mitotic tether, indicating this interaction is insufficient for E2 association with mitotic chromosomes. RNAi-induced depletion of ChlR1 significantly reduced E2 localization to mitotic chromosomes. These studies provide compelling evidence that ChlR1 association is required for loading the papillomavirus E2 protein onto mitotic chromosomes and represents a kinetochore-independent mechanism for viral genome maintenance and segregation.

Animals↗
PubMed2004

Role of the putative structural protein Sed1p in mitochondrial genome maintenance.

The nuclear gene MIP1 encodes the mitochondrial DNA polymerase responsible for replicating the mitochondrial genome in Saccharomyces cerevisiae. A number of other factors involved in replicating and segregating the mitochondrial genome are yet to be identified. Here, we report that a bacterial two-hybrid screen using the mitochondrial polymerase, Mip1p, as bait identified the yeast protein Sed1p. Sed1p is a cell surface protein highly expressed in the stationary phase. We find that several modified forms of Sed1p are expressed and the largest of these forms interacts with the mitochondrial polymerase in vitro. Deletion of SED1 causes a 3.5-fold increase in the rate of mitochondrial DNA point mutations as well as a 4.3-fold increase in the rate of loss of respiration. In contrast, we see no change in the rate of nuclear point mutations indicating the specific role of Sed1p function in mitochondrial genome stability. Indirect immunofluorescence analysis of Sed1p localization shows that Sed1p is targeted to the mitochondria. Moreover, Sed1p is detected in purified mitochondrial fractions and the localization to the mitochondria of the largest modified form is insensitive to the action of proteinase K. Deletion of the sed1 gene results in a reduction in the quantity of Mip1p and also affects the levels of a mitochondrially-expressed protein, Cox3p. Our results point towards a role for Sed1p in mitochondrial genome maintenance.

Base Sequence↗
PubMed2001

Genome maintenance mechanisms for preventing cancer.

The early notion that cancer is caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. During the past decade, knowledge about the mechanisms by which genes erode and the molecular machinery designed to counteract this time-dependent genetic degeneration has increased markedly. At the same time, it has become apparent that inherited or acquired deficiencies in genome maintenance systems contribute significantly to the onset of cancer. This review summarizes the main DNA caretaking systems and their impact on genome stability and carcinogenesis.

Animals↗
PubMed2003

Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance.

A Cre/lox-conditional mouse line was generated to evaluate the role of ATR in checkpoint responses to ionizing radiation (IR) and stalled DNA replication. We demonstrate that after IR treatment, ATR and ATM each contribute to early delay in M-phase entry but that ATR regulates a majority of the late phase (2-9 h post-IR). Double deletion of ATR and ATM eliminates nearly all IR-induced delay, indicating that ATR and ATM cooperate in the IR-induced G2/M-phase checkpoint. In contrast to the IR-induced checkpoint, checkpoint delay in response to stalled DNA replication is intact in ATR knockout cells and ATR/ATM and ATR/p53 double-knockout cells. The DNA replication checkpoint remains intact in ATR knockout cells even though the checkpoint-stimulated inhibitory phosphorylation of Cdc2 on T14/Y15 and activating phosphorylation of the Chk1 kinase no longer occur. Thus, incomplete DNA replication in mammalian cells can prevent M-phase entry independently of ATR and inhibitory phosphorylation of Cdc2. When DNA replication inhibitors are removed, ATR knockout cells proceed to mitosis but do so with chromosome breaks, indicating that ATR provides a key genome maintenance function in S phase.

Animals↗
PubMed2004

A genotoxic screen: rapid analysis of cellular dose-response to a wide range of agents that either damage DNA or alter genome maintenance pathways.

SNP analysis has come to the forefront of genomics since the mouse and human genomes have been sequenced. High throughput functional screens are necessary to evaluate these sequence databases. Described here is a genotoxic screen: a rapid method that determines the cellular dose-response to a wide range of agents that either damage DNA or alter basic cellular pathways important for maintaining genomic integrity. Importantly, a single person utilizing standard tissue culture equipment may perform these assays composed of 20 agents that attack genomic integrity or maintenance at many different levels. Thus, a small lab may perform this screen to determine the integrity of a wide range of DNA repair, chromatin metabolism, and response pathways without the limitations of investigator bias. A genotoxic screen will be useful when analyzing cells with either known genetic alterations (generated directly by the investigator or derived from individuals with known mutations) or unknown genetic alterations (cells with spontaneous mutations such as cancer-derived cells). Screening many genotoxins at one time will aid in determining the biological importance of these altered genes. Here we show the dose-response curves of mouse embryonic stem (ES) cells and HeLa cells exposed to 20 genotoxic agents. ES cells were chosen since they are amenable to genetic alteration by the investigator. HeLa cells were chosen since they were derived from cancer and are commonly used. Comparing the dose-response curves of these two cell lines show their relative sensitivity to these agents and helps define their genotoxic profile. As a part of phenomics, a large genotoxic profile database for cancer-derived cells, when integrated with other databases such as expression profiles and comparative genomic hybridization, may aid in maximizing the effectiveness of developing anti-cancer protocols.

Alkylating Agents↗
PubMed2003

RecQ helicases: multiple roles in genome maintenance.

The RecQ helicases are highly conserved in evolution and are required for maintaining genome stability in all organisms. In humans, loss of RecQ helicase function is associated with predisposition to cancer and/or premature ageing. Recent data show that RecQ helicases have several roles during S phase of the cell cycle, ranging from facilitating the resumption of DNA synthesis at sites of replication fork breakdown to resolving structures during the process of homologous recombination.

Animals↗
PubMed1997

Cytoplasmic tyrosyl-tRNA synthetase rescues the defect in mitochondrial genome maintenance caused by the nuclear mutation mgm104-1 in the yeast Saccharomyces cerevisiae.

The yeast nuclear mutation mgm104-1, which leads to slow growth on glucose medium and temperature-sensitive (ts) loss of mitochondrial DNA (mtDNA), has been identified by screening a collection of temperature-sensitive mutants on glycerol medium. A nuclear gene was isolated from a genomic DNA library by complementation of the mgm104-1 allele and was found to be identical to TTS1, which encodes the cytoplasmic tyrosyl-tRNA synthetase required for cytoplasmic protein synthesis. A gene disruption in a diploid strain demonstrated that the TTS1 gene is essential for cell viability. The lack of mutations in TTS1 in the mgm104-1 mutant indicates that TTS1 and MGM104 are different genes. The ability to rescue the mgm104-1 phenotype with a single additional copy of TTS1 suggests that TTS1 has an additional function that is directly or indirectly involved in the maintenance of the mitochondrial genome.

Base Sequence↗