Genome analysis of DNA repair genes in the alpha proteobacterium <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Background  

The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria.     

Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background  

Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair.     

Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

Background  

Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci.     

Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases

Background

Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G₁/S and G₂/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation.

Results

Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined.

Conclusion

Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of enhanced BER activities was found in irradiated cells arrested in G2 phase.     

Characterization of the meningococcal DNA glycosylase Fpg involved in base excision repair

Background  

Neisseria meningitidis, the causative agent of meningococcal disease, is exposed to high levels of reactive oxygen species inside its exclusive human host. The DNA glycosylase Fpg of the base excision repair pathway (BER) is a central player in the correction of oxidative DNA damage. This study aimed at characterizing the meningococcal Fpg and its role in DNA repair.     

A single hydrophobic cleft in the <Emphasis Type="Italic">Escherichia coli</Emphasis> processivity clamp is sufficient to support cell viability and DNA damage-induced mutagenesis <Emphasis Type="Italic">in vivo</Emphasis>

Background  

The ubiquitous family of DnaN sliding processivity clamp proteins plays essential roles in DNA replication, DNA repair, and cell cycle progression, in part by managing the actions of the different proteins involved in these processes. Interactions of the homodimeric Escherichia coli β clamp with its known partners involves multiple surfaces, including a hydrophobic cleft located near the C-terminus of each clamp protomer.     

Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations

Background  

DNA polymerase ε (Pol ε) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect.     

Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase

Background  

Efficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood.     

The alpha/beta fold uracil DNA glycosylases: a common origin with diverse fates

Background  

Uracil DNA glycosylases (UDGs) are major repair enzymes that protect DNA from mutational damage caused by uracil incorporated as a result of a polymerase error or deamination of cytosine. Four distinct families of UDGs have been identified, which show very limited sequence similarity to each other, although two of them have been shown to possess the same structural fold. The structural and evolutionary relationships between the rest of the UDGs remain uncertain.     

Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

Background  

DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage.     

Decline of nucleotide excision repair capacity in aging <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

Caenorhabditis elegans is an important model for the study of DNA damage and repair related processes such as aging, neurodegeneration, and carcinogenesis. However, DNA repair is poorly characterized in this organism. We adapted a quantitative polymerase chain reaction assay to characterize repair of DNA damage induced by ultraviolet type C (UVC) radiation in C. elegans, and then tested whether DNA repair rates were affected by age in adults.     

Pioglitazone retrieves hepatic antioxidant DNA repair in a mice model of high fat diet

Background  

Pioglitazone was reported to improve hepatic steatosis and necroinflammation in human studies. To investigate whether the hepato-protective effect of pioglitazone was associated with an improvement of antioxidant defense mechanism, oxidative DNA damage and repair activity were determined in a high fat diet model. Male C57BL/6 mice were respectively fed with a 30% fat diet, the same diet with pioglitazone 100 mg/kg/day, or a chow diet as control for 8 weeks. Tissue oxidative stress was indicated by malondialdehyde concentration. Oxidative DNA damage was detected by immunohistochemical 8-oxoG staining. Enzymatic antioxidant defense was detected by the real-time PCR of superoxide dismutase (Sod1, Sod2) and DNA glycosylase (Ogg1, MutY). Oxidative DNA repair was detected by immunohistochemical staining and western blotting of OGG1 expression.     

The PD-(D/E)XK superfamily revisited: identification of new members among proteins involved in DNA metabolism and functional predictions for domains of (hitherto) unknown function

Background  

The PD-(D/E)XK nuclease superfamily, initially identified in type II restriction endonucleases and later in many enzymes involved in DNA recombination and repair, is one of the most challenging targets for protein sequence analysis and structure prediction. Typically, the sequence similarity between these proteins is so low, that most of the relationships between known members of the PD-(D/E)XK superfamily were identified only after the corresponding structures were determined experimentally. Thus, it is tempting to speculate that among the uncharacterized protein families, there are potential nucleases that remain to be discovered, but their identification requires more sensitive tools than traditional PSI-BLAST searches.     

ICRPfinder: a fast pattern design algorithm for coding sequences and its application in finding potential restriction enzyme recognition sites

Background  

Restriction enzymes can produce easily definable segments from DNA sequences by using a variety of cut patterns. There are, however, no software tools that can aid in gene building -- that is, modifying wild-type DNA sequences to express the same wild-type amino acid sequences but with enhanced codons, specific cut sites, unique post-translational modifications, and other engineered-in components for recombinant applications. A fast DNA pattern design algorithm, ICRPfinder, is provided in this paper and applied to find or create potential recognition sites in target coding sequences.     

Novel splice variants associated with one of the zebrafish <Emphasis Type="Italic">dnmt3</Emphasis>genes

Background  

DNA methylation and the methyltransferases are known to be important in vertebrate development and this may be particularly true for the Dnmt3 family of enzymes because they are thought to be the de novo methyltransferases. Mammals have three Dnmt3 genes; Dnmt3a, Dnmt3b, and Dnmt3L, two of which encode active enzymes and one of which produces an inactive but necessary cofactor. However, due to multiple promoter use and alternative splicing there are actually a number of dnmt3 isoforms present. Six different dnmt3 genes have recently been identified in zebrafish.     

Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol ε and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors

Background  

Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved.     

Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases

Background  

Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments.