Molecular mechanisms of carcinogenesis: the role of systems of DNA repair

The initiation step of the carcinogenic process consists in an alteration of genes playing a central role in the cellular life. The next steps of promotion and progression result from anomalies in the response to growth factors, to hormones and/or from the action of tumor promotors leading to cellular hyperplasia. This process generally leads to genetic instability of the initiated cell which in turn allows selection of malignant and invasive clones. The production of DNA damage by physical or chemical agents is dose-dependent. The error-free enzymatic repair processes including excision resynthesis of base damage or of altered nucleotides allow the restitution of intact DNA. The error-prone repair systems permit survival in association with transmissible alterations (genes and chromosomal mutations). Absence of repair leads to cytotoxicity, programmed cell death or disruption of cell cycle control leading to a pretumoral state. The major role played by mutations in the initiation of carcinogenesis is evidenced by the existence of genetic syndromes associated to hypersensitivity to genotoxic agents, defects in DNA repair capacity, anomalies in the expression of certain genes (including the tumor suppressor p53 gene, etc.) and an elevated predisposition to cancer. Xeroderma pigmentosum which is defective in excision-repair, ataxia telangiectasia and Fanconi anemia which are associated to anomalies in DNA recombination and the familial type of colon cancer HPNCP due to inefficient mismatch repair constitute paradigm for this fundamental notion. Alterations in the capacity to rejoin radiation induced DNA strand breaks appears to be associated to over-reactions to radiotherapy of cancer patients. Also the predisposition to develop secondary thyroid tumors following treatment of a primary cancer in childhood seems to involve the same defect. The existence in the general population of heterozygotes for such DNA repair genes should be taken into account for risk evaluation to therapeutic and environmental exposures.     

The role of 3'-5' exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance

In the D171G/D230A mutant generated at conserved aspartate residues in the Exo1 and Exo2 sites of the 3'-5' exonuclease domain of the yeast mitochondrial DNA (mtDNA) polymerase (pol-gamma), the mitochondrial genome is unstable and the frequency of mtDNA point mutations is 1500 times higher than in the wild-type strain and 10 times higher than in single substitution mutants. The 10(4)-fold decrease in the 3'-5' exonuclease activity of the purified mtDNA polymerase is associated with mismatch extension and high rates of base misincorporation. Processivity of the purified polymerase on primed single-stranded DNA is decreased and the Km for dNTP is increased. The sequencing of mtDNA point mutations in the wild-type strain and in proofreading and mismatch-repair deficient mutants shows that mismatch repair contributes to elimination of the transitions while exonucleolytic proofreading preferentially repairs transversions, and more specifically A to T (or T to A) transversions. However, even in the wild-type strain, A to T (or T to A) transversions are the most frequent substitutions, suggesting that they are imperfectly repaired. The combination of both mismatch repair and proofreading deficiencies elicits a mitochondrial error catastrophe. These data show that the faithful replication of yeast mtDNA requires both exonucleolytic proofreading and mismatch repair.     

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.     

Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase.     

The effect of nicotine on DNA repair in adult myocytes

Recent findings have demonstrated that terminally differentiated adult ventricular myocytes are capable of repairing DNA that has been damaged by exposure to oxygen free radicals. Despite the potential importance of DNA repair in cells that may survive many decades after injury, little is known about the mechanisms or regulation of repair. Since tobacco use has a well-defined role in the epidemiology and pathophysiology of heart disease, we tested the effects of nicotine on repair of free radical damaged plasmids by whole-cell protein extracts from adult myocytes. Exposure to a concentration of 25 microM nicotine increased incorporation of (32P)dCTP into damaged plasmids by 16%, and 50 or 100 microM nicotine increased incorporation by 32%. Nicotine did not alter the rate or amount of poly (ADP-ribose) on the major protein acceptor of molecular weight 113-116 kDa. Inhibition of DNA polymerase activity with pyridoxal 5'-phosphate revealed greater plasmid degradation in the presence of nicotine. We conclude that nicotine enhances DNA degradation and the increased repair is a consequence of this greater degradation.     

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.     

The role immaturity in human development.

The possibility that infants' and young children's immature behaviors and cognitions are sometimes adaptive is explored and interpreted in terms of evolutionary theory. It is argued that developmental immaturity had an adaptive role in evolution and continues to have an adaptive role in human development. The role of developmental retardation in human evolution is discussed, followed by an examination of the relation between humans' extended childhood and brain plasticity. Behavioral neoteny, as exemplified by play, is examined, as are some potentially adaptive aspects of infants' perception and cognition that limit the amount of information they can process. Aspects of immature cognition during early childhood that may have some contemporaneous adaptive value are also discussed. It is proposed that viewing immaturity as sometimes adaptive to the developing child alters how children and their development are viewed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The role of function in conceptual development.

40 3-, 40 5-, and 40 7-yr-olds were administered a free-sorting task, a directed sorting task, a verbal labeling task, and a conversation task using 4 natural concepts: cup, scissors, money, and musical instrument. Results reveal that almost all Ss had a knowledge of the correct function for cup and scissors, and all but the 3-yr-olds knew similar information for money and musical instrument. However, none of the Ss used all of the knowledge they possessed to guide behavior in the free-sorting task. The 5- and 7-yr-olds could use their knowledge when cued in the directed sort and when forced to use it in the verbal labeling task. In comparison, the 3-yr-olds were relatively unable to use their knowledge to guide behavior in any of these 3 tasks. Inferences are drawn about both the structure of children's concepts and how that structure changes with development. (French abstract) (25 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The role of homeobox genes in limb development

Intensive study of the development of the vertebrate limb has led to a conceptual framework for understanding the specification of a limb primordium, the outgrowth of those cells and their organization and differentiation into a functional appendage. During the past few years, a number of homeobox-containing genes have been identified that are likely to play controlling roles in each of these events.     

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 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.     

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.     

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.     

The role of single-stranded DNA in Flp-mediated strand exchange

The Flp recognition target site contains two inverted 13-base pair (bp) Flp binding sequences that surround an 8-bp core region. Flp recombinase has been shown to carry out strand ligation independently of its ability to execute strand cleavage. Using a synthetic activated DNA substrate bearing a 3'-phosphotyrosine group, we have developed an assay to measure strand exchange by Flp proteins. We have shown that wild-type Flp protein was able to catalyze strand exchange using DNA substrates containing 8-bp duplex core sequences. Mutant Flp proteins that are defective in either DNA bending or DNA cleavage were also impaired in their abilities to carry out strand exchange. The inability of these mutant proteins to execute strand exchange could be overcome by providing a DNA substrate containing a single-stranded core sequence. This single-stranded core sequence could be as small as 3 nucleotides. Full activity of mutant Flp proteins in strand exchange was observed when both partner DNAs contained an 8-nucleotide single-stranded core region. Using suicide substrates, we showed that single-stranded DNA is also important for strand exchange reactions where Flp-mediated strand cleavage is required. These results suggest that the ability of Flp to induce DNA bending and strand cleavage may be crucial for strand exchange. We propose that both DNA bending and strand cleavage may be required to separate the strands of the core region and that single-stranded DNA in the core region might be an intermediate in Flp-mediated DNA recombination.     

The fission yeast UVDR DNA repair pathway is inducible

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.