Cisplatin resistance and regulation of DNA repair in cAMP-dependent protein kinase mutants

Drug resistance in cancer poses a major problem to the success of chemotherapy. Increased resistance to the DNA-damaging chemotherapeutic drug cisplatin may be associated with a variety of factors including decreased drug accumulation, increased intracellular levels of thiols, and increased DNA repair. We have found that mutants of the Chinese hamster ovary (CHO) and the mouse adrenocortical carcinoma Y1 cells harboring a defective regulatory subunit (RI) of the cAMP-dependent protein kinase (PKA) exhibited increased resistance to cisplatin. These mutants are cross-resistant to other DNA-damaging chemotherapeutic agents, including bleomycin and melphalan. In addition, wild-type CHO cells transfected with and overexpressing the yeast phosphodiesterase gene or a dominant mutant Rl alpha subunit gene also displayed similar increased resistance to cisplatin. However, mutants with altered catalytic (C) subunits showed a sensitivity to cisplatin similar to the wild-type cells. Further analysis by gel shift assay using cisplatin-damaged DNA as probes and nuclear extracts derived from the Rl subunit mutants showed increased binding of nuclear factor(s) to the damaged DNA. In addition, a host cell reactivation assay of DNA repair, using a cisplatin-damaged reporter plasmid, detected enhanced capacity for repair of DNA lesions in the PKA mutants. These results suggest that DNA repair may be increased in the PKA mutants. We speculate that functional inactivation of PKA may result in increased DNA repair and the acquisition of resistance to DNA-damaging anticancer drugs in cancer.     

DNA replication, repair, and repair tests

The rate of inhibition and recovery of DNA synthesis can be used in a rapid assay system to detect genotoxic potentials of chemicals. Also, the observation that an agent stimulates DNA repair in a test system indicates its ability to cause damage in DNA. Different experimental approaches to the study of repair synthesis are discussed.     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase alpha is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase delta functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase zeta, for mutagenesis. The function of DNA polymerase epsilon in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase delta or epsilon suffices for the repair of UV-induced damage. The role of DNA polymerase beta in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase delta appears to fulfill that function.     

Conserved domains in DNA repair proteins and evolution of repair systems

A detailed analysis of protein domains involved in DNA repair was performed by comparing the sequences of the repair proteins from two well-studied model organisms, the bacterium Escherichia coli and yeast Saccharomyces cerevisiae, to the entire sets of protein sequences encoded in completely sequenced genomes of bacteria, archaea and eukaryotes. Previously uncharacterized conserved domains involved in repair were identified, namely four families of nucleases and a family of eukaryotic repair proteins related to the proliferating cell nuclear antigen. In addition, a number of previously undetected occurrences of known conserved domains were detected; for example, a modified helix-hairpin-helix nucleic acid-binding domain in archaeal and eukaryotic RecA homologs. There is a limited repertoire of conserved domains, primarily ATPases and nucleases, nucleic acid-binding domains and adaptor (protein-protein interaction) domains that comprise the repair machinery in all cells, but very few of the repair proteins are represented by orthologs with conserved domain architecture across the three superkingdoms of life. Both the external environment of an organism and the internal environment of the cell, such as the chromatin superstructure in eukaryotes, seem to have a profound effect on the layout of the repair systems. Another factor that apparently has made a major contribution to the composition of the repair machinery is horizontal gene transfer, particularly the invasion of eukaryotic genomes by organellar genes, but also a number of likely transfer events between bacteria and archaea. Several additional general trends in the evolution of repair proteins were noticed; in particular, multiple, independent fusions of helicase and nuclease domains, and independent inactivation of enzymatic domains that apparently retain adaptor or regulatory functions.     

DNA repair in higher plants

Numerous studies have demonstrated a requirement in plants for repair of DNA damage arising from either intrinsic or extrinsic sources. Investigations also have revealed a capacity for repair of certain types of DNA damage, and conversely, identified mutants apparently defective in such repair. This article provides a concise overview of nuclear DNA repair mechanisms in higher plants, particularly those processes concerned with the repair of UV-induced lesions, and includes surveys of UV-sensitive mutants and genes implicated in DNA repair.     

DNA repair functions in heterologous cells

Our genetic information is constantly challenged by exposure to endogenous and exogenous DNA-damaging agents, by DNA polymerase errors, and thereby inherent instability of the DNA molecule itself. The integrity of our genetic information is maintained by numerous DNA repair pathways, and the importance of these pathways is underscored by their remarkable structural and functional conservation across the evolutionary spectrum. Because of the highly conserved nature of DNA repair, the enzymes involved in this crucial function are often able to function in heterologous cells; as an example, the E. coli Ada DNA repair methyltransferase functions efficiently in yeast, in cultured rodent and human cells, in transgenic mice, and in ex vivo-modified mouse bone marrow cells. The heterologous expression of DNA repair functions has not only been used as a powerful cloning strategy, but also for the exploration of the biological and biochemical features of numerous enzymes involved in DNA repair pathways. In this review we highlight examples where the expression of DNA repair enzymes in heterologous cells was used to address fundamental questions about DNA repair processes in many different organisms.     

Mammalian base excision repair and DNA polymerase beta

OBJECTIVE: To describe six dogs with congenital abnormalities involving the portal vein, caudal vena cava, or both. ANIMALS: Six client-owned dogs with congenital interruption of the portal vein or the caudal vena cava, or both. METHODS: Portal vein and caudal vena cava anatomy was evaluated by contrast radiography and visualization at surgery. Vascular casts or plastinated specimens were obtained in three animals. RESULTS: Portal blood shunted into the caudal vena cava in four dogs and the left hepatic vein in one. Two of these five dogs also had interruption of the caudal vena cava with continuation as azygous vein, as did an additional dog, in which the portal vein was normally formed. Portal vein interruption was present in 5 of 74 (6.8%) dogs with congenital portosystemic shunts evaluated at the Veterinary Teaching Hospital during the study period. CONCLUSIONS: Serious malformations of the abdominal veins were present in more than 1 in 20 dogs with single congenital portosystemic shunts. CLINICAL RELEVANCE: Veterinarians involved in diagnosis and surgery for portosystemic shunts should be aware of these potential malformations, and portal vein continuity should be evaluated in all dogs before attempting shunt attenuation.     

H-NS regulates DNA repair in Shigella

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30 degrees C and nonfunctional at 37 to 40 degrees C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40 degrees C postirradiation, shows up to 30-fold higher survival than when incubated at 30 degrees C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40 degrees C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40 degrees C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40 degrees C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.     

Replication of O6-methylguanine-containing DNA by repair and replicative DNA polymerases

The biological consequences of O6-methylguanine (m6G) in DNA are well recognized. When template m6G is encountered by DNA polymerases, replication is hindered and trans-lesion replication results in the preferential incorporation of dTMP opposite template m6G. Thus, unrepaired m6G in DNA is both cytotoxic and mutagenic. Yet, cell lines tolerant to m6G in DNA have been isolated, which indicates that some cellular DNA polymerases may replicate m6G-containing DNA with reasonable efficiency. Previous reports suggested that mammalian pol beta could not replicate m6G-containing DNA, but we find that pol beta can catalyze trans-lesion replication; however, the lesion must reside in the optimal context for pol beta activity, single- or short nucleotide gapped substrates. Primed single-stranded DNA templates, with or without template m6G, were poor substrates for pol beta as reported in earlier studies. In contrast, trans-lesion replication by bacteriophage T4 DNA polymerase was observed for primed single-stranded DNA templates. Replication of m6G-containing DNA by T4 DNA polymerase required the gp45 accessory protein that clamps the polymerase to the DNA template. The rate-limiting step in replicating m6G-containing DNAs by both DNA polymerases tested was incorporation of dTMP across from the lesion.     

Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways

Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes.     

Allelic losses and DNA methylation at DNA mismatch repair loci in sporadic colorectal cancer

Normal and tumor DNA samples of 35 patients with sporadic colorectal carcinoma were analyzed for microsatellite alterations at 12 markers linked to mismatch repair loci: hMLH1, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2. Remarkably, no correlation was observed between the replication error phenotype (RER+) and allelic losses at these loci. Hemizygous deletions, seen in 6/35 (17%) informative cases at hMLH1, 4/27 (15%) at hMSH2/hMSH6 and 6/34 (18%) at hMSH3, were rarely found in RER+ tumors. Since mismatch repair protein components act in molecular complexes of defined stoichiometry we propose that hemizygous deletion of the corresponding loci may be involved in colorectal tumorigenesis through defects in cellular functions other than replication error correction. The analysis of the methylation status of the promoter region of hMLH1 revealed that methylation might be an important mechanism of this locus inactivation in RER+ sporadic colorectal cancer.     

Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage

Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging. Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood. By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1. A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2). Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria. Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria. hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria. hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria. In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells. These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.     

Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair

Eukaryotic DNA polymerase beta (pol beta) can catalyze DNA synthesis during base excision DNA repair. It is shown here that pol beta also catalyzes release of 5'-terminal deoxyribose phosphate (dRP) residues from incised apurinic-apyrimidinic sites, which are common intermediate products in base excision repair. The catalytic domain for this activity resides within an amino-terminal 8-kilodalton fragment of pol beta, which comprises a distinct structural domain of the enzyme. Magnesium is required for the release of dRP from double-stranded DNA but not from a single-stranded oligonucleotide. Analysis of the released products indicates that the excision reaction occurs by beta-elimination rather than hydrolysis.