The relative roles of three DNA repair pathways in preventing Caenorhabditis elegans mutation accumulation

Mutation is a central biological process whose rates and spectra are influenced by a variety of complex and interacting forces. Although DNA repair pathways are generally known to play key roles in maintaining genetic stability, much remains to be understood about the relative roles of different pathways in preventing the accumulation of mutations and the extent of heterogeneity in pathway-specific repair efficiencies across different genomic regions. In this study we examine mutation processes in base excision repair-deficient (nth-1) and nucleotide excision repair-deficient (xpa-1) Caenorhabditis elegans mutation-accumulation (MA) lines across 24 regions of the genome and compare our observations to previous data from mismatch repair-deficient (msh-2 and msh-6) and wild-type (N2) MA lines. Drastic variation in both average and locus-specific mutation rates, ranging two orders of magnitude for the latter, was detected among the four sets of repair-deficient MA lines. Our work provides critical insights into the relative roles of three DNA repair pathways in preventing C. elegans mutation accumulation and provides evidence for the presence of pathway-specific DNA repair territories in the C. elegans genome.     

Strand-specific processing of 8-oxoguanine by the human mismatch repair pathway: inefficient removal of 8-oxoguanine paired with adenine or cytosine

Genomic DNA and its precursors are susceptible to oxidation during aerobic cellular metabolism, and at least five distinct repair activities target a single common lesion, 7,8-dihydro-8-oxoguanine (8-oxoG). The human mismatch repair (MMR) pathway, which has been implicated in an apoptotic response to covalent DNA damage, is likely to encounter 8-oxoG in both the parental and daughter strand during replication. Here, we show that lesions containing 8-oxoG paired with adenine or cytosine, which are most likely to arise during replication, are not efficiently processed by the mismatch repair system. Lesions containing 8-oxoG paired with thymine or guanine, which are unlikely to arise, are excised in an MSH2/MSH6-dependent manner as effectively as the corresponding mismatches when placed in a context that reflects the daughter strand during replication. Using a newly developed assay based on methylation sensitivity, we characterized strand-excision events opposite 8-oxoG situated to reflect placement in the parental strand. Lesions that efficiently trigger strand excision and resynthesis (8-oxoG paired with thymine or guanine) result in adenine or cytosine insertion opposite 8-oxoG. These latter pairings are poor substrates for further action by mismatch repair, but precursors for alternative pathways with non-mutagenic outcomes. We suggest that the lesions most likely to be encountered by the human mismatch repair pathway during replication, 8-oxoG.A or 8-oxoG.C, are likely to escape processing in either strand by this system. Taken together, these data suggest that the human mismatch repair pathway is not a major contributor to removal of misincorporated 8-oxoG, nor is it likely to trigger repeated attempts at lesion processing.     

The C. elegans glycosyltransferase BUS-8 has two distinct and essential roles in epidermal morphogenesis

Ventral enclosure in Caenorhabditis elegans involves migration of epidermal cells over a neuroblast substrate and subsequent adhesion at the ventral midline. Organisation of the neuroblast layer by ephrins and their receptors is essential for this migration. We show that bus-8, which encodes a predicted glycosyltransferase, is essential for embryonic enclosure and acts in or with ephrin signalling to mediate neuroblast organisation and to permit epidermal migration. BUS-8 acts non-cell-autonomously in this process, and likely modifies an extracellular regulator of ephrin signalling and cell organisation. Weak and cold-sensitive alleles of bus-8 show that the gene has a separate and distinct post-embryonic role, being essential for epidermal integrity and production of the cuticle surface. This disorganisation of the epidermis and cuticle layers causes increased drug sensitivity, which could aid the growing use of C. elegans in drug screening and chemical genomics. The viable mutants are also resistant to infection by the pathogen Microbacterium nematophilum, due to failure of the bacterium to bind to the host surface. The two separate essential roles of BUS-8 in epidermal morphogenesis add to our growing understanding of the widespread importance of glycobiology in development.     

Acetylation of human 8-oxoguanine-DNA glycosylase by p300 and its role in 8-oxoguanine repair in vivo

The human 8-oxoguanine-DNA glycosylase 1 (OGG1) is the major DNA glycosylase responsible for repair of 7,8-dihydro-8-oxoguanine (8-oxoG) and ring-opened fapyguanine, critical mutagenic DNA lesions that are induced by reactive oxygen species. Here we show that OGG1 is acetylated by p300 in vivo predominantly at Lys338/Lys341. About 20% of OGG1 is present in acetylated form in HeLa cells. Acetylation significantly increases OGG1's activity in vitro in the presence of AP-endonuclease by reducing its affinity for the abasic (AP) site product. The enhanced rate of repair of 8-oxoG in the genome by wild-type OGG1 but not the K338R/K341R mutant, ectopically expressed in oxidatively stressed OGG1-null mouse embryonic fibroblasts, suggests that acetylation increases OGG1 activity in vivo. At the same time, acetylation of OGG1 was increased by about 2.5-fold after oxidative stress with no change at the polypeptide level. OGG1 interacts with class I histone deacetylases, which may be responsible for its deacetylation. Based on these results, we propose a novel regulatory function of OGG1 acetylation in repair of its substrates in oxidatively stressed cells.     

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10–13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approximately −6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (ΔG° approximately −3.3 kcal/mol) together with other specific interactions (ΔG° approximately −0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the k_cat term instead; the rate increases by 6–7 orders of magnitude for cognate DNA as compared with non-cognate one. The k_cat values for substrates of different sequences correlate with the DNA twist, while the K_M values correlate with ΔG° of the DNA fragments surrounding the lesion (position from −6 to +6). The functions for predicting the K_M and k_cat values for different sequences containing oxoG were found.     

Repair of 8-oxoguanine in Saccharomyces cerevisiae: interplay of DNA repair and replication mechanisms

8-Oxo-7,8-dihydroguanine (8-oxoG) is produced abundantly in DNA exposed to free radicals and reactive oxygen species. The biological relevance of 8-oxoG has been unveiled by the study of two mutator genes in Escherichia coli, fpg, and mutY. Both genes code for DNA N-glycosylases that cooperate to prevent the mutagenic effects of 8-oxoG in DNA. In Saccharomyces cerevisiae, the OGG1 gene encodes a DNA N-glycosylase/AP lyase, which is the functional homologue of the bacterial fpg gene product. The inactivation of OGG1 in yeast creates a mutator phenotype that is specific for the generation of GC to TA transversions. In yeast, nucleotide excision repair (NER) also contributes to the release of 8-oxoG in damaged DNA. Furthermore, mismatch repair (MMR) mediated by MSH2/MSH6/MLH1 plays a major role in the prevention of the mutagenic effect of 8-oxoG. Indeed, MMR acts as the functional homologue of the MutY protein of E. coli, excising the adenine incorporated opposite 8-oxoG. Finally, the efficient and accurate replication of 8-oxoG by the yeast DNA polymerase η also prevents 8-oxoG-induced mutagenesis. The aim of this review is to summarize recent literature dealing with the replication and repair of 8-oxoG in Saccharomyces cerevisiae, which can be used as a paradigm for DNA repair in eukaryotes.     

Modeling oncogenic translocations: distinct roles for double-strand break repair pathways in translocation formation in mammalian cells

Reciprocal chromosomal translocations are implicated in the etiology of many tumors, including leukemias, lymphomas, and sarcomas. DNA double-strand breaks (DSBs) caused by various cellular processes and exogenous agents are thought to be responsible for the generation of most translocations. Mammalian cells have multiple pathways for repairing DSBs in the chromosomes: non-homologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA), which is a specialized pathway involving sequence repeats. In this review, we summarize the various reporters that have been used to examine the potential for each of these DSB repair pathways to mediate translocation formation in mammalian cells. This approach has demonstrated that NHEJ is very proficient at mediating translocation formation, while HR is not because of crossover suppression. Although SSA can efficiently mediate translocations between identical repeats, its contribution to translocation formation is likely very limited because of sequence divergence between repetitive elements in the genome.     

8-hydroxydeoxyguanosine causes death of human leukemia cells deficient in 8-oxoguanine glycosylase 1 activity by inducing apoptosis

Our previous study showed that KG-1, a human acute leukemia cell line, has mutational loss of 8-oxoguanine (8-hydroxyguanine; oh(8)Gua) glycosylase 1 (OGG1) activity and that its viability is severely affected by 8-hydroxydeoxyguanosine (8-oxodeoxyguanosine; oh(8)dG). In the present study, the nature of the killing action of oh(8)dG on KG-1 was investigated. Signs observed in oh(8)dG-treated KG-1 cells indicated that death was due to apoptosis, as demonstrated by: increased sub-G(1) hypodiploid (apoptotic) cells, DNA fragmentation, and apoptotic body formation; loss of mitochondrial transmembrane potential, the release of cytochrome c from mitochondria into the cytosol, and the down-regulation of bcl-2; and the activation of caspases 8, 9, and 3, and the efficient inhibition of the apoptotic process by caspases inhibitors. This apoptosis appears not to be associated with Fas/Fas ligand because the expressions of these proteins were unchanged. Apoptotic KG-1 cells showed a high concentration of oh(8)Gua in DNA. Moreover, the increased concentration of oh(8)Gua in DNA, and the apoptotic process were not suppressed by the antioxidant, N-acetylcysteine, and thus the process is independent of reactive oxygen species. Of the 18 cancer cell lines treated with oh(8)dG, 3 cell lines (H9, CEM-CM3, and Molt-4) were found to be committed to apoptosis, and all of these showed very low OGG1 activity and a marked increase in the concentration of oh(8)Gua in DNA. These observations indicate that in addition to its mutagenic action, oh(8)Gua in DNA disturbs cell viability by inducing apoptosis.     

Characterization of chimeric enzymes constructed between two distinct alpha-amylase cDNAs from cultured rice cells.

Cultured cells of rice (Oryza sativa cv Sasanishiki) produce two alpha-amylase isozymes, AMY-I and AMY-III. Using a bacterial expression system, eight chimeric genes constructed with various combination of AMY-I and AMY-III cDNA fragments were expressed, and each recombinant chimeric protein was characterized. Four of the eight recombinant enzymes having region c (one of the four regions having unconserved base sequences between AMY-I and AMY-III cDNAs) of AMY-I showed the same enzyme characteristics as that of native AMY-I, which had high temperature optimum at 50 degrees C. The other four chimeric proteins carrying region c of AMY-III showed the AMY-III type characteristics, which were a low temperature optimum at 25 degrees C and susceptibility to a higher maltooligosaccharide (G17) substrate. The unconserved region c is involved in the decision of the characteristic of AMY-I or AMY-III.     

The presence of two mitochondrial DNA topoisomerases in human acute leukemia cells

We have undertaken a study of DNA topoisomerases in mitochondria from human acute leukemia cells. Two activities have been detected in these organelles. One of the enzymes is presumably a type II topoisomerase, i.e., in ATP-dependent reactions it can catenate closed circular plasmid DNA, and decatenate closed circular kinetoplast DNA. A second topoisomerase is presumably a type I enzyme since, it can relax positive as well as negative supercoils in an ATP-independent reaction, it is unable to catenate plasmid DNA or decatenate kinetoplast DNA, and it is inhibited, rather than stimulated, by ATP.     

Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites.

The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.     

Backbone dynamics of DNA containing 8-oxoguanine: importance for substrate recognition by base excision repair glycosylases.

Except for the functional groups sited within the major or minor grooves, the bases of B-DNA are quite protected from the external environment. Enzymes that modify the bases often "flip out" the target into an extrahelical position before the chemistry step is carried out. Examples of this mechanism are the base excision repair glycosylases and the restriction enzyme methylases. The question arises about the mechanism of substrate recognition for these enzymes and how closely it is linked to the base flipping step. Molecular dynamics simulations (AMBER, PME electrostatics) of fully solvated, cation neutralized, DNA sequences containing 8-oxoguanine (8OG) and of appropriate normal (control) DNAs have been carried out. The dynamics trajectories were analyzed to identify those properties of the DNA structure in the vicinity of the altered base, or its dynamics, that could contribute to molecular discrimination between substrate and non-substrate DNA sites. The results predict that the FPG enzyme should flip out the cytosine base paired with the scissile 8OG, not the target base itself.     

The mammalian XRCC genes: their roles in DNA repair and genetic stability

Analysis of the XRCC genes has played an important part in understanding mammalian DNA repair processes, especially those involved in double-strand break (DSB) repair. Most of these genes were identified through their ability to correct DNA damage hypersensitivity in rodent cell lines, and they represent components of several different repair pathways including base-excision repair, non-homologous end joining, and homologous recombination. We document the phenotypic effects of mutation of the XRCC genes, and the current state of our knowledge of their functions. In addition to their continuing importance in discovering mechanisms of DNA repair, analysis of the XRCC genes is making a substantial contribution to the understanding of specific human disorders, including cancer.     

Identification of early postmitotic cells in distinct embryonic sites and their possible roles in morphogenesis

Differential proliferation within defined embryonic anlage is likely to play a major role in morphogenesis. We have identified cell populations in the avian embryo that begin exiting the cell cycle as early as the 25-somite stage. These include first the floor plate and then the roof plate of the neural tube, cells that constitute the lamina terminalis and the diencephalic-mesencephalic junction of the developing brain. Outside the nervous system, the central portion of the notochord contains early postmitotic cells. In the heart, such cells will populate the epimyocardium at the level of the truncus arteriosus exclusively and the endocardial cushions that serve as an anchor for the growing intracardial septa. Surprisingly, the endoderm at the level of the prospective midgut is composed of post-mitotic progenitors. These cells are later found both in the caudal portion of the duodenum and in derivatives adjacent to the umbilical region of the primitive midgut. The possible implications of this early, localized withdrawal from the cell cycle to morphogenetic events and lineage segregation are discussed.     

On the presence of two non-specific nucleotide-sugar-hydrolysing enzymes in rat liver.

Evidence is presented for the occurrence of two different non-specific nucleotide-sugar hydrolases in rat liver and other rat tissues. These two enzymes (I and II) were separated by chromatography on a 5'-AMP-aminohexyl-Sepharose column. Enzyme I is most probably identical with phosphodiesterase I (EC 3.1.4.1). Enzyme II appeared to be identical with an enzyme described in literature as 'CMP-sialic acid hydrolase' [Kean & Bighouse (1974) J. Biol. Chem. 249, 7813-7823], since almost all activity with CMP-N-acetylneuraminate as substrate was recovered in this enzyme fraction. CMP-N-acetylneuraminate was a poor substrate for Enzyme I, whereas deoxythymidine-5'-p-nitrophenyl phosphate and all nucleoside-diphosphosugars tested were good substrates for both Enzyme I and II. Therefore it is suggested that CMP-N-acetylneuraminate is used as an additional substrate to discriminate between the activities of Enzyme I and II in homogenates or membrane preparations. The various substrates appeared to be competitive inhibitors of each other, suggesting that, in each enzyme preparation, only one enzyme is responsible for the hydrolysis of the various substrates. The dissimilar properties of the two enzymes are substantiated by studying the subunit molecular masses (Enzyme I, 125 kDa; Enzyme II, 50-55 kDa), the sensitivity towards Triton X-100, Sarkosyl and sodium dodecyl sulphate and towards trypsin treatment. It is discussed whether the alpha-N-acetylglucosamine phosphodiesterase described by Varki & Kornfeld [(1981) J. Biol. Chem. 256, 9937-9943] is identical with one of the nucleotide-sugar hydrolases described here.     

The presence of two GDP-L-fucose: glycoproteine fucosyltransferases in human serum

Two soluble fucosyltransferases have been demonstrated in human serum. One enzyme transfers l-fucose from GDP-l-fucose to the terminal galactose residues of lactose, N-acetyllactosamine, and sialidase-treated α₁-acid glycoprotein, to form the blood group H determinant, α-l-fucosyl-(1 → 2)-β-d-galactosyl-R. The second enzyme transfers fucose to the terminal N-acetylglucosamine residue of sialidase-, β-galactosidase-treated α₁-acid glycoprotein. Serum from a donor with the rare “Bombay” O_h blood group (genotype hh) cannot transfer fucose to terminal galactose residues but has normal levels of the enzyme acting on sialidase-, β-galactosidase-treated α₁-acid glycoprotein. This observation, as well as mixed substrate experiments, demonstrate that the two fucosyltransferase activities are due to two separate enzymes. The GDP-l-fucose:galactoside fucosyltransferase has a pH optimum of 5.5 and the following K_m values: lactose, 31 mm; N-acetyllactosamine, 7.5 mm; sialidase-treated α₁-acid glycoprotein, 6.4 mm. The GDP-l-fucose: N-acetylglucosaminide fucosyltransferase has a pH optimum of 5.0 and a K_m for sialidase-, β-galactosidase-treated α₁-acid glycoprotein of 1.2 mm. The serum GDP-l-fucose: N-acetylglucosaminide fucosyltransferase is distinct from the blood group Lewis-dependent enzyme in milk since the serum enzyme is present in serum from Le (a-b-)donors and since the Le-dependent fucosyltransferase could not be demonstrated in serum from donors carrying the Le gene.