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PubMed2005

The Elg1 replication factor C-like complex: a novel guardian of genome stability.

The remarkable stability of the eukaryotic genome is achieved by the activity of many overlapping surveillance and repair mechanism. Two protein complexes with resemblance to replication factor C (RFC) have been recently described, that play important roles in maintaining the stability of the genome. These RFC-like complexes (RLCs) share four common subunits (Rfc2-5) and each carry a unique large subunit (Rad24 or Ctf18) replacing the Rfc1 subunit of the replication complex. Work in several laboratories has recently uncovered a novel yeast gene, ELG1, which seems to play a central role in keeping the genome stable. elg1 mutants exhibit increased rates of spontaneous recombination and gross chromosomal rearrangements during vegetative growth. In addition, they lose chromosomes at an enhanced rate, show hyper-transposition of natural repeated elements and exhibit elongated telomeres. The Elg1 protein also associates with the Rfc2-5 subunits of replication factor C (RFC) to form a third RFC-like complex (RLC). Genetic and biochemical data indicate that the Elg1, Ctf18 and Rad24 RLCs work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses. ELG1 is evolutionarily conserved and may play an important role in preventing the onset of cancer in humans. The Elg1 function is thus clearly required for maintaining genome stability during normal growth, and its absence has severe genetic consequences.

Carrier Proteins↗
PubMed1994

Long-term genome stability and minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-, immortalized human uroepithelial cells.

Parameters of genome instability and morphological alterations associated with cell transformation were studied in an isogeneic set of clonal human uroepithelial cell (HUC) lines immortalized by the human papilloma virus 16 (HPV16) E6 and/or E7 gene(s). HPV16 E6 binds p53, leading to rapid degradation of p53, whereas E7 binds and alters pRb and other proteins. We report that two independent E7-immortalized HUC lines showed minimal phenotypic or genotypic alterations, except that both lines contained amplification of 20q DNA sequences and a greater polyploidization at an early passage. The E7-immortalized HUC line resembled normal HUC lines, except that they failed to senesce. In contrast, the E6-immortalized HUC lines were morphologically altered, contained numerous random chromosome aberrations, and showed unstable evolving karyotypes with passage in culture. No amplified DNA sequences were detected in E6-immortalized HUC lines. Instead, clonal losses of chromosome regions (i.e., -3p, -6q, -9p), putatively containing tumor suppressor or senescence genes, accompanied the E6-HUC immortalization event. E6-immortalized HUC lines showed transformed phenotypes similar to E6/E7-HUC lines. The difference in genome stability between E6- and E7-immortalized HUC was highly significant statistically (p-value < 10(-6). Thus, the HPV16 E7 gene led to HUC immortalization by a pathway that blocked cellular senescence, but did not disrupt genome stability. These results implicate p53 loss, but not pRb alteration, in genome destabilization.

Base Sequence↗
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↗
PubMed1995

Genomic stability and wild-type p53 function of lymphoblastoid cells with germ-line p53 mutation.

Increased cancer risk associated with germ-line p53 mutation was linked to a deficit in the ability to maintain genomic stability. Accordingly, normal fibroblasts from cancer-prone individuals accumulate genomic aberrations with concomitant loss of wild-type p53 allele during in vitro culture. We tested whether such changes also occur in EBV-immortalized lymphoblastoid cells. Both normal and p53 germ-line mutant lymphoblastoid cells maintained functional p53 and genomic stability during long term in vitro culture. These unexpected differences between fibroblastic and lymphoblastic cells suggest that phenotypic expression of p53 deficiency is cell type specific. This could contribute to selective tissular localization of tumours observed in patients with Li-Fraumeni syndrome despite the presence of a mutant p53 allele in all cells.

Alleles↗
PubMed2005

Bloom syndrome ortholog HIM-6 maintains genomic stability in C. elegans.

Bloom syndrome is caused by mutation of the Bloom helicase (BLM), a member of the RecQ helicase family. Loss of BLM function results in genomic instability that causes a high incidence of cancer. It has been demonstrated that BLM is important for maintaining genomic stability by playing a role in DNA recombination and repair; however, the exact function of BLM is not clearly understood. To determine the mechanism by which BLM controls genomic stability in vivo, we examined the phenotypes caused by mutation of the C. elegans BLM helicase ortholog, HIM-6. We find that the loss of HIM-6 leads to genomic instability as evidenced by an increased number of genomic insertions and deletions, which results in visible random mutant phenotypes. In addition to the mutator phenotype, him-6 mutants have a low brood size, a high incidence of males, a shortened life span, and an increased amount of germ line apoptosis. Upon exposure to high temperature, him-6 mutants that are serially passed become sterile demonstrating a mortal germ line phenotype. Our data suggest a model in which loss of HIM-6 results in genomic instability due to an increased number of DNA lesions, which either cannot be repaired and/or are introduced by low fidelity recombination events. The increased level of genomic instability that leads to him-6(ok412) mutants having a shortened life span.

Animals↗
PubMedDate not supplied

[Dynamic component of maintaining genomic stability in murine bone marrow cells after chronic low-intensity irradiation lasting one year].

For analysis of a dynamic component state of the system of maintenance of genome stability, which represent a condition of its expression (first of all, genes of the control of phases of cell cycle, the DNA repair and redox systems) after a long chronic exposure to a small dose, the activity of replicative, reparative DNA synthesis, DNA damage as well as oxyradical content in the bone marrow cells of mice (critical for radiation effects mammalian system) after 1 year radiation exposure to a dose 63.7 cGy (0.17 cGy/day) were studied. The considerable enhancement of replicative and reparative DNA synthesis activity by 67% (p = 0.0033) and 60% (p = 0.000004) accordingly in relation to the control and some, but statistically not significant (p = 0.149) tendency to increase (by 30%) the content of a superoxide anion-radical were established. Strong and highly significant correlation (r = 0.8681; P = 0.99975) between DNA damage and O2-. content in bone marrow cells of the irradiated mices, which indicate the large DNA damage by oxiradicals, probably, due to the loss of a part of structural proteins and conformation changes in expression sites of a chromatin, were detected. The obtained results interpreted as representing the change of a dynamic component state of a system of maintenance of genome stability, the epigenic transfer of that to descendants of the irradiated cells can be the cause for formation and maintenance of radiation-induced genome instability.

Animals↗
PubMed2006

A unique role of the DNA fragmentation factor in maintaining genomic stability.

DNA fragmentation is a hallmark of apoptosis (programmed cell death). However, the biological function of apoptotic DNA fragmentation remains unclear. Here, we show that DNA fragmentation factor plays an important role for maintaining genomic stability. Inhibition or loss of the DNA fragmentation factor (DFF)/caspase-activated DNase (CAD), whose nuclease activity is responsible for digesting genomic DNA during apoptosis, led to significant increases in spontaneous or induced gene mutations, gene amplifications, and chromosomal instability in primary mouse cells and transformed human cell lines. The mechanism underlying genetic instability in DFF/CAD-deficient cells, at least in part, involves a small but significant elevation in the survival of cells exposed to ionizing radiation, suggesting that apoptotic DNA fragmentation factor contributes to genomic stability by ensuring the removal of cells that have suffered DNA damage. In support of this hypothesis are the observations of increased cellular transformation of mouse embryonic cells from the DFF/CAD-null mice and significantly enhanced susceptibility to radiation-induced carcinogenesis in these mice. These data, in combination with published reports on the existence of tumor-specific gene mutations/deletions in the DFF/CAD genes in human cancer samples, suggest that apoptotic DNA fragmentation factor is required for the maintenance of genetic stability and may play a role in tumor suppression.

Animals↗
PubMed2003

Caloric restriction promotes genomic stability by induction of base excision repair and reversal of its age-related decline.

Caloric restriction is a potent experimental manipulation that extends mean and maximum life span and delays the onset and progression of tumors in laboratory rodents. While caloric restriction (CR) clearly protects the genome from deleterious damage, the mechanism by which genomic stability is achieved remains unclear. We provide evidence that CR promotes genomic stability by increasing DNA repair capacity, specifically base excision repair (BER). CR completely reverses the age-related decline in BER capacity (P<0.01) in all tissues tested (brain, liver, spleen and testes) providing aged, CR animals with the BER phenotype of young, ad libitum-fed animals. This CR-induced reversal of the aged BER phenotype is accompanied by a reversal in the age-related decline in DNA polymerase beta (beta-pol), a rate-limiting enzyme in the BER pathway. CR significantly reversed the age-related loss of beta-pol protein levels (P<0.01), mRNA levels (P<0.01) and enzyme activity (P<0.01) in all tissues tested. Additionally, in young (4-6-month-old) CR animals a significant up-regulation in BER capacity, beta-pol protein and beta-pol mRNA is observed (P<0.01), demonstrating an early effect of CR that may provide insight in distinguishing the anti-tumor from the anti-aging effects of CR. This up-regulation in BER by caloric restriction in young animals corresponds to increased protection from carcinogen exposure, as mutation frequency is significantly reduced in CR animals exposed to either DMS or 2-nitropropane (2-NP) (P<0.01). Overall the data suggest an important biological consequence of moderate BER up-regulation and provides support for the hormesis theory of caloric restriction.

Aging↗
PubMed2006

Cotranscriptional processes and their influence on genome stability.

Numerous studies support the idea that the complex process of gene expression is composed of multiple highly coordinated and integrated steps. While such an extensive coupling ensures the efficiency and accuracy of each step during the gene expression pathway, recent studies have suggested an evolutionarily conserved function for cotranscriptional processes in the maintenance of genome stability. Specifically, such processes prevent a detrimental effect of nascent transcripts on the integrity of the genome. Here we describe studies indicating that nascent transcripts can rehybridize with template DNA, and that this can lead to DNA strand breaks and rearrangements. We present an overview of the diverse mechanisms that different species employ to keep nascent RNA away from DNA during transcription. We also discuss possible mechanisms by which nascent transcripts impact genome stability, as well as the possibility that transcription-induced genomic instability may contribute to disease.

Biological Evolution↗
PubMed2003

CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing.

To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of 'licensing proteins' at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle. Here we show that the CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 (refs 2, 3) is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. We propose that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1.

Animals↗
PubMed1999

Chromosomal aberrations in PARP(-/-) mice: genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA.

Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP(-/-) mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP(-/-)mice and immortalized PARP(-/-)fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP(-/-) cells stably transfected with PARP cDNA [PARP(-/-)(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP(-/-) cells, p53 expression was partially restored in PARP(-/-) (+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene Rb-1 in PARP(-/-) mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene Jun was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.

Animals↗
PubMed2003

Telomeres in the chicken: genome stability and chromosome ends.

Telomeres are the complex nucleoprotein structures at the termini of linear chromosomes. Telomeric DNA consists of a highly conserved hexanucleotide arranged in tandem repeats. Telomerase, a ribonucleoprotein of the reverse transcriptase family, specifies the sequence of telomeric DNA and maintains telomere array length. Numerous studies in model organisms established the significance of telomere structure and function in regulating genome stability, cellular aging, and oncogenesis. Our overall research objectives are to understand the organization of the telomere arrays in chicken in the context of the unusual organization and specialized features of this higher vertebrate genome (which include a compact genome, numerous microchromosomes, and high recombination rate) and to elucidate the role telomeres play in genome stability impacting cell function and life span. Recent studies found that the chicken genome contains three overlapping size classes of telomere arrays that differ in location and age-related stability: Class I 0.5 to 10 kb, Class II 10 to 40 kb, and Class III 40 kb to 2 Mb. Some notable features of chicken telomere biology are that the chicken genome contains ten times more telomeric DNA than the human genome and the Class III telomere arrays are the largest described for any vertebrate species. In vivo, chicken telomeres (Class II) shorten in an age-related fashion and telomerase activity is high in early stage embryos and developing organs but down-regulates during late embryogenesis or postnatally in most somatic tissues. In vitro, chicken cells down-regulate telomerase activity unless transformed. Knowledge of chicken telomere biology contributes information relevant to present and future biotechnology applications of chickens in vivo and chicken cells in vitro.

Aging↗
PubMed2006

Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability.

Helicases are molecular motor proteins that couple the hydrolysis of NTP to nucleic acid unwinding. The growing number of DNA helicases implicated in human disease suggests that their vital specialized roles in cellular pathways are important for the maintenance of genome stability. In particular, mutations in genes of the RecQ family of DNA helicases result in chromosomal instability diseases of premature aging and/or cancer predisposition. We will discuss the mechanisms of RecQ helicases in pathways of DNA metabolism. A review of RecQ helicases from bacteria to human reveals their importance in genomic stability by their participation with other proteins to resolve DNA replication and recombination intermediates. In the light of their known catalytic activities and protein interactions, proposed models for RecQ function will be summarized with an emphasis on how this distinct class of enzymes functions in chromosomal stability maintenance and prevention of human disease and cancer.

Adenosine Triphosphatases↗
PubMed2006

Rac1 GTPase regulates cell genomic stability and senescence.

The Rho family small GTPase Rac1 has been shown to play multiple roles in cell regulation, including actin cytoskeleton organization, transcriptional activation, microtubule dynamics, and endocytosis. Here, we report a novel role of Rac1 in regulating genomic stability and cell senescence. We observed in primary mouse embryonic fibroblasts that deletion of rac1 by gene targeting, as well as expression of the constitutively active Rac1 mutant L61Rac1, led to decreased cell growth that was associated with altered cell cycle progression at both G(1)/S and G(2)/M phases, increased apoptosis, and premature senescence. The senescence induction by either loss or gain of Rac1 activity was due at least in part to an increase in cellular reactive oxygen species (ROS). rac1 gene deletion caused a compensatory up-regulation of a closely related family member, Rac3, in mouse embryonic fibroblasts, the activity of which induced ROS production independently of Rac1. Furthermore, the Rac1-regulated ROS production and senescence correlated with the extent of DNA damage in the Rac1(-/-) and L61Rac1 cells. Treatment of these cells with a ROS inhibitor inhibited phospho-H2AX-positive nuclear focus formation. Finally, phospho-Ser(15) p53 was significantly increased in L61Rac1 and Rac1(-/-) cells, and genetic deletion of p53 from these cells readily reversed the senescence phenotype, indicating that Rac1 is functionally dependent on p53 in regulating cell senescence. Taken together, our results show that Rac1 activity serves as a regulator of cell senescence through modulation of cellular ROS, genomic stability, and p53 activity.

Animals↗
PubMed2001

A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability.

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA(+) class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.

Adenosine Triphosphatases↗
PubMed2006

The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability.

The BRCA1 and BRCA2 proteins are important in maintaining genomic stability by promoting efficient and precise repair of double-strand breaks. The main role of BRCA2 appears to involve regulating the function of RAD51 in the repair by homologous recombination. BRCA1 has a broader role upstream of BRCA2, participating in various cellular processes in response to DNA damage. The DNA repair defect associated with mutations in BRCA1 or BRCA2 could be exploited to develop new targeted therapeutic approaches for cancer occurring in mutation carriers.

BRCA1 Protein↗
PubMed2003

Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability.

Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors. The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition. Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability.

Chromatin Assembly Factor-1↗
PubMed2005

Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex.

Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination. On their own, mutations in RMI1 result in phenotypes that mimic those of sgs1 or top3 strains including slow growth, hyperrecombination, DNA damage sensitivity, and reduced sporulation. And like top3 strains, most rmi1 phenotypes are suppressed by mutations in SGS1. We show that Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast and that these proteins interact directly in a recombinant system. The Rmi1-Top3 complex is stable in the absence of the Sgs1 helicase, but the loss of either Rmi1 or Top3 in yeast compromises its partner's interaction with Sgs1. Biochemical studies demonstrate that recombinant Rmi1 is a structure-specific DNA binding protein with a preference for cruciform structures. We propose that the DNA binding specificity of Rmi1 plays a role in targeting Sgs1-Top3 to appropriate substrates.

Amino Acid Sequence↗