Prevent Genome Instability Research Articles

Ku70 prevents genome instability resulting from heterozygosity of the telomerase RNA component in a vertebrate tumour line

Telomere repeat sequences are added to linear chromosome ends by telomerase, an enzyme comprising a reverse transcriptase (TERT) and an RNA template component (TR). We aimed to investigate TR in the DT40 B-cell tumour line using gene targeting, but were unable to generate TR nulls, suggesting a requirement for TR in DT40 proliferation. Disruption of one TR allele reduced telomerase activity and caused a progressive decline in telomere and G-strand overhang length. We then examined the interactions between TR and cellular DNA double-strand break (DSB) repair. Deletion in TR +/− cells of the gene encoding the non-homologous end-joining protein, Ku70, caused rapid loss of G-strand overhangs. Ku70 −/− TR +/− cells proliferated more slowly than either single mutant and showed frequent mitotic aberrations. Activation of the DNA damage response was observed in TR-deficient cells and was exacerbated by Ku deficiency, although frequent telomeric DNA damage signals were not observed until late passages. This activation of the DNA damage response was suppressed by deletion of Rad54, a key homologous recombination gene. These findings suggest that Ku and telomerase cooperate to block homologous recombination from acting on telomeres.

Chromatin Responses to DNA Damage

Proper repair of DNA damage is critical in preventing genome instability and carcinogenesis. While the role of ATP‐dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double strand break (DSB) in yeast. Our findings reveal a new role of ATP‐dependent chromatin remodeling in nuclear processes and suggest that an ATP‐dependent chromatin remodeling complex can read a DNA repair histone code. We have also found recently that a subunit of the INO80 complex is phosphorylated in an ATM/ATR‐dependent fashion in response to DNA damage, and the phosphorylation events modulate DNA checkpoint pathways. Thus, chromatin responses to DNA damages involve complex changes in both histones and complexes that regulate chromatin.

Xeroderma Pigmentosum: A Glimpse into Nucleotide Excision Repair, Genetic Instability, and Cancer

Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by extreme sensitivity to sunlight and severe predisposition to UV-induced skin cancer. Seven genes, ranging from XPA to XPG, are defective in XP. These genes are important components of the nucleotide excision repair (NER) system, which removes DNA damage induced by solar radiation, thereby preventing genome instability and carcinogenesis. In addition, XPV patients are defective in a translesion synthesis activity specialized in bypassing UV-induced lesions, and share symptoms with other XP patients. This review will focus on the evidence that elucidates the link between defective NER, genetic instability, and oncogenesis.

DDB1 Maintains Genome Integrity through Regulation of Cdt1

DDB1, a component of a Cul4A ubiquitin ligase complex, promotes nucleotide excision repair (NER) and regulates DNA replication. We have investigated the role of human DDB1 in maintaining genome stability. DDB1-depleted cells accumulate DNA double-strand breaks in widely dispersed regions throughout the genome and have activated ATM and ATR cell cycle checkpoints. Depletion of Cul4A yields similar phenotypes, indicating that an E3 ligase function of DDB1 is important for genome maintenance. In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved. One substrate of DDB1-Cul4A that is crucial for preventing genome instability is Cdt1. DDB1-depleted cells exhibit increased levels of Cdt1 protein and rereplication, despite containing other Cdt1 regulatory mechanisms. The rereplication, accumulation of DNA damage, and activation of checkpoint responses in DDB1-depleted cells require entry into S phase and are partially, but not completely, suppressed by codepletion of Cdt1. Therefore, DDB1 prevents DNA lesions from accumulating in replicating human cells, in part by regulating Cdt1 degradation.

The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells

Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instability.

Bloom syndrome, genomic instability and cancer: the SOS-like hypothesis

Bloom syndrome (BS) displays one of the strongest known correlations between chromosomal instability and an increased risk of malignancy at an early age. The prevention of genomic instability and cancer depends on a complex network of pathways induced in response to DNA damage and stalled replication forks, including cell-cycle checkpoints, DNA repair, and apoptosis. Several studies have demonstrated that BLM is involved in the cellular response to DNA damage and stalled replication forks. BLM interacts physically and functionally with several proteins involved in the maintenance of genome integrity and BLM is redistributed and/or phosphorylated in response to several genotoxic stresses. The data concerning the relationship between BLM and these cellular pathways are summarized and the role of BLM in the rescue of arrested replication forks is discussed. Moreover, I speculate that BLM deficiency is lethal, and that BLM-deficient cells escaping apoptotic death do so by constitutively inducing a bacterial SOS-like response including the induction of alternative replication pathway(s) dependent on recombination, contributing to the mutator and hyper-Rec phenotypes characteristic of BS cells. This mechanism may be dependent on the RAD51 gene family, and involved in carcinogenesis in the general population.

Exo1 Processes Stalled Replication Forks and Counteracts Fork Reversal in Checkpoint-Defective Cells

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.

Radiation and the cell cycle, revisited.

The cell cycle has been inextricably linked to the cellular response to radiation for many years. However, it is only in the past decade that the concept of a coordinated DNA damage response integrating damage recognition, cell cycle checkpoints and DNA repair has begun to be elucidated. The ATM protein is emerging as a key orchestrator of the damage response activating a wide variety of effectors involved in cell cycle arrest and DNA repair to elicit a concerted effort to prevent genome instability caused by unwanted changes in DNA sequence. The key proteins involved in cell cycle checkpoints in different phases of the cell cycle, and their interaction, is a fertile and rapidly developing area of research. This review summarizes the current state of knowledge of cellular checkpoints in response to radiation-induced double-strand breaks in mammalian cells and how this impacts on radiosensitivity.

BRCA1 and BRCA2 in Breast Cancer Predisposition and Recombination Control

Hereditary predisposition to breast and ovarian cancer is determined in large part by loss-of-function mutations in one of two genes, BRCA1 and BRCA2 . Early discoveries that the two genes function in the control of homologous recombination and the prevention of genomic instability have been strongly supported by subsequent work. Our aim here is to highlight new advances in the study of BRCA1 and BRCA2 , and to place these advances in the context of existing knowledge.

Genome hypermethylation in Pinus silvestris of Chernobyl—a mechanism for radiation adaptation?

Adaptation is a complex process by which populations of organisms respond to long-term environmental stresses by permanent genetic change. Here we present data from the natural “open-field” radiation adaptation experiment after the Chernobyl accident and provide the first evidence of the involvement of epigenetic changes in adaptation of a eukaryote-Scots pine ( Pinus silvestris), to chronic radiation exposure. We have evaluated global genome methylation of control and radiation-exposed pine trees using a method based on cleavage by a methylation-sensitive HpaII restriction endonuclease that leaves a 5′ guanine overhang and subsequent single nucleotide extension with labeled [ 3 H ] dCTP. We have found that genomic DNA of exposed pine trees was considerably hypermethylated. Moreover, hypermethylation appeared to be dependent upon the radiation dose absorbed by the trees. Such hypermethylation may be viewed as a defense strategy of plants that prevents genome instability and reshuffling of the hereditary material, allowing survival in an extreme environment. Further studies are clearly needed to analyze in detail the involvement of DNA methylation and other epigenetic mechanisms in the complex process of radiation stress and adaptive response.

Werner's syndrome protein is phosphorylated in an ATR/ATM-dependent manner following replication arrest and DNA damage induced during the S phase of the cell cycle.

Werner's syndrome (WS) is an autosomal recessive disorder, characterized at the cellular level by genomic instability in the form of variegated translocation mosaicism and extensive deletions. Individuals with WS prematurely develop multiple age-related pathologies and exhibit increased incidence of cancer. WRN, the gene defective in WS, encodes a 160-kDa protein (WRN), which has 3'-5'exonuclease, DNA helicase and DNA-dependent ATPase activities. WRN-defective cells are hypersensitive to certain genotoxic agents that cause replication arrest and/or double-strand breaks at the replication fork, suggesting a pivotal role for WRN in the protection of the integrity of the genoma during the DNA replication process. Here, we show that WRN is phosphorylated through an ATR/ATM dependent pathway in response to replication blockage. However, we provide evidence that WRN phosphorylation is not essential for its subnuclear relocalization after replication arrest. Finally, we show that WRN and ATR colocalize after replication fork arrest, suggesting that WRN and the ATR kinase collaborate to prevent genome instability during the S phase.

Saccharomyces cerevisiae MGS1 is essential in strains deficient in the RAD6-dependent DNA damage tolerance pathway.

Saccharomyces cerevisiae Mgs1 protein, which possesses DNA-dependent ATPase and single strand DNA annealing activities, plays a role in maintaining genomic stability. We found that mgs1 is synthetic lethal with rad6 and exhibits a synergistic growth defect with rad18 and rad5, which are members of the RAD6 epistasis group important for tolerance of DNA damage during DNA replication. The mgs1 mutant is not sensitive to DNA-damaging agents, but the mgs1 rad5 double mutant has increased sensitivity to hydroxyurea and a greatly increased spontaneous mutation rate. Growth defects of mgs1 rad18 double mutants are suppressed by a mutation in SRS2, encoding a DNA helicase, or by overexpression of Rad52. More over, mgs1 mutation suppresses the temperature sensitivity of mutants in POL3, encoding DNA polymerase delta. mgs1 also suppresses the growth defect of a pol3 mutant caused by expression of Escherichia coli RuvC, a bacterial Holliday junction resolvase. These findings suggest that Mgs1 is essential for preventing genome instability caused by replication fork arrest in cells deficient in the RAD6 pathway and may modulate replication fork movement catalyzed by yeast polymerase delta.

High-copy-number expression of Sub2p, a member of the RNA helicase superfamily, suppresses hpr1-mediated genomic instability.

We report on a novel role for a pre-mRNA splicing component in genome stability. The Hpr1 protein, a component of an RNA polymerase II complex and required for transcription elongation, is also required for genome stability. Deletion of HPR1 results in a 1,000-fold increase in genome instability, detected as direct-repeat instability. This instability can be suppressed by the high-copy-number SUB2 gene, which is the Saccharomyces cerevisiae homologue of the human splicing factor hUAP56. Although SUB2 is essential, conditional alleles grown at the permissive temperature complement the essential function of SUB2 yet reveal nonessential phenotypes. These studies have uncovered a role for SUB2 in preventing genome instability. The genomic instability observed in sub2 mutants can be suppressed by high-copy-number HPR1. A deletion mutant of CDC73, a component of a PolII complex, is also unstable for direct repeats. This too is suppressed by high-copy-number SUB2. Thus, defects in both the transcriptional machinery and the pre-mRNA splicing machinery can be sources of genome instability. The ability of a pre-mRNA splicing factor to suppress the hyperrecombination phenotype of a defective PolII complex raises the possibility of integrating transcription, RNA processing, and genome stability or a second role for SUB2.

The 3'-->5' exonuclease of DNA polymerase delta can substitute for the 5' flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.

Many DNA polymerases (Pol) have an intrinsic 3'-->5' exonuclease (Exo) activity which corrects polymerase errors and prevents mutations. We describe a role of the 3'-->5' Exo of Pol delta as a supplement or backup for the Rad27/Fen1 5' flap endonuclease. A yeast rad27 null allele was lethal in combination with Pol delta mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol delta. The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol delta Exo I and Exo II motifs. However, rad27-p Pol delta Exo III double mutants were viable. They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability. Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5' flaps in the lagging strand. These results suggest that the 3'-->5' Exo activity of Pol delta is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand.