Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences (original) (raw)
Related papers
Molecular Cell, 2013
To improve replication fidelity, mismatch repair (MMR) must detect non-Watson-Crick base pairs and direct their repair to the nascent DNA strand. Eukaryotic MMR in vitro requires pre-existing strand discontinuities for initiation; consequently, it has been postulated that MMR in vivo initiates at Okazaki fragment termini in the lagging strand and at nicks generated in the leading strand by the mismatch-activated MLH1/PMS2 endonuclease. We now show that a single ribonucleotide in the vicinity of a mismatch can act as an initiation site for MMR in human cell extracts and that MMR activation in this system is dependent on RNase H2. As loss of RNase H2 in S.cerevisiae results in a mild MMR defect that is reflected in increased mutagenesis, MMR in vivo might also initiate at RNase H2-generated nicks. We therefore propose that ribonucleotides misincoporated during DNA replication serve as physiological markers of the nascent DNA strand.
Repair of a Mismatch Is Influenced by the Base Composition of the Surrounding Nucleotide Sequence
Genetics, 1987
Heteroduplexes with single base pair mismatches of known sequence were prepared by annealing separated strands of bacteriophage λ DNA and used to transfect Escherichia coli. A series of transition (G:T and A:C) and transversion (G:A and C:T) mismatches located throughout most of the bacteriophage λ cI gene has been examined. The results suggest that the transition mismatches are generally better repaired than the transversion mismatches and that, at least for the transversion mismatches studied, repair efficiency increases with increasing G:C content in the neighboring nucleotide sequence. This specificity of the E. coli mismatch repair system can account, in part, for the similar frequencies of base substitution mutations throughout the E. coli genome.
Biochemistry, 1995
In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment. Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA). To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions. Purified DNA polymerase 6 or E, with DNA ligase, was then used to form the repair patch. DNA polymerase 6 could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles. The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products. DNA polymerase E, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles. Addition of replication .protein A changed the situation dramatically, and synthesis by polymerase E became dependent on both PCNA and replication factor C. A combination of DNA polymerase E , PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches. Exposure of genomic DNA to physical or chemical mutagens leads to the formation of a range of bulky lesions that are efficiently removed by nucleotide excision repair. This repair process requires the interaction of many gene products with damaged DNA, some of which also participate in DNA replication and transcription (Weeda & Hoeijmakers, 1993; Aboussekhra & Wood, 1994). A coordinated sequence of events begins with the recognition, incision, and excision of the damage from the DNA. Many of the genes and gene products involved in this stage of the nucleotide excision repair process in mammalian cells have been identified by studying the inherited, autosomally recessive disease xeroderma pigmentosum (XP). The defective nucleotide excision repair pathway in XP cells causes an increased sensitivity to UV light, and can lead to a large increase in the incidence of sunlight-induced skin cancer (Cleaver & Kraemer, 1989). Normally, damage is excised as part of an oligomer about 30 nucleotides long (Svoboda et al., 1993). A repair patch of about 30 nucleotides is then formed in vivo (Cleaver et al., 1991) and in vitro (Hansson et al., 1989; Shivji et al., 1992). The repair synthesis stage is known to require the DNA polymerase accessory factor PCNA. Fractionation of
Patch length of localized repair events: role of DNA polymerase I in mutY-dependent mismatch repair
Journal of Bacteriology
In vivo experiments with heteroduplex lambda genomes show that the MutY mismatch repair system of Escherichia coli defines an average repair tract that is shorter than 27 nucleotides and longer than 9 nucleotides and extends 3' from the corrected adenine. The phenotype of a mutant defective in DNA polymerase I shows that this enzyme plays a significant, though not an essential, role in the in vivo repair of apurinic sites generated by this system. Evidence is presented that in the absence of polymerase I the repair tracts are modestly longer than in the poLA+ extending in the 5' direction from the corrected adenine, suggesting a role for another DNA polymerase.
Nucleotide excision repair DNA synthesis by overexpressed DNA polymeraseß: a new error-prone pathway
The FASEB Journal
The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases ␦ and/or participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase  exhibited a fiveto sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol  in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol  is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol  in cancer cells could perturb the well-defined specific functions of DNA polymerases during errorfree DNA transactions.-Canitrot, Y., Hoffmann, J.-S., Calsou, P., Hayakawa, H., Salles, B., Cazaux, C. Nucleotide excision repair DNA synthesis by excess DNA polymerase : a potential source of genetic instability in cancer cells. FASEB J. 14, 1765-1774 (2000)
Recognition of DNA alterations by the mismatch repair system
Biochemical Journal, 1999
Misincorporation of non-complementary bases by DNA polymerases is a major source of the occurrence of promutagenic base-pairing errors during DNA replication or repair. Base-base mismatches or loops of extra bases can arise which, if left unrepaired, will generate point or frameshift mutations respectively. To counteract this mutagenic potential, organisms have developed a number of elaborate surveillance and repair strategies which cooperate to maintain the integrity of their genomes. An important replication-associated correction function is provided by the post-replicative mismatch repair system. This system is highly conserved among species and appears to be the major pathway for strand-specific elimination of base-base mispairs and short insertion\deletion loops (IDLs), not only during DNA replication, but also in intermediates of homologous recombination. The efficiency of repair of different base-pairing errors in the DNA varies, and appears to depend on multiple factors, such as the physical structure of the mismatch and sequence
The EMBO Journal, 1997
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 β-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T ϾG ϾA) 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.
Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols b and l are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol b, to a greater extent than Pol l can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol band l-deficient cell extracts suggest that Pol b levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. | www.nature.com/naturecommunications *The meaning of the kinetic parameters K m , k cat and k cat /K m and their calculations are described in the Methods section. Values are the means of two independent estimates ±s.d. w f inc , relative incorporation frequencies for the different nucleotide pairs, defined as the ratio of the respective k cat /K m values. Figure 4 | Incorporation of rNMPs opposite 8-oxo-G inhibits DNA repair and is reduced in the absence of DNA polymerase b. (a) Time course of the excision products accumulation generated by hOGG1 in the presence of a 8-oxo-G:dC (empty circles) or 8-oxo-G:rC (filled triangles) base pairs. The k app values refer to the apparent rates for the exponential phase. Values are the mean of three independent replicates, error bars represent ±s.d. A representative experiment is shown in (b) Quantification of the excision products generated by MutYH in the presence of a 8-oxo-G:dA (grey bars) or 8-oxo-G:rA (black bars) mismatch. Values are the mean of three independent replicates as the one presented in , error bars represent ± s.d. The P values were calculated by two-tailed Student's t-test. (c) Increasing amounts of the different cell extracts were titrated in the presence of the 1 nt-gap template bearing an 8-oxo-G and 200 mM rCTP. Lanes 13 and 14, control reactions in the absence of extracts. (d) The rCMP incorporation activity (expressed as pmols of rCTP incorporated opposite 8-oxo-G per mg of proteins of each extract) was normalized to the total DNA polymerase activity (expressed as pmols of dCMP incorporated opposite undamaged dG per mg of proteins of each extract). Values are the mean of three independent replicates like the one shown in c, error bars represent ±s.d. The P values were calculated by two-tailed Student's t-test.
DNA Mismatch Repair Systems: Mechanisms and Applications in Biotechnology
Biotechnology and Genetic Engineering Reviews, 1993
Mismatch repair systems: Mechanisms and specificities The detection of mispaired or unpaired bases in DNA is a central element of mutation avoidance (Radman et al., 1979) genetic recombination (Jones, Wagner and Radman, 1987a) and speciation (Rayssiguier, Thaler and Radman, 1989). Mispaired or unpaired bases may arise as a result of replication errors, strand exchange between homologous but non-identical sequences or deamination of 5-methyl-cytosine (5-meC) to thymine. Both replication errors and deaminations of 5-meC are changes in DNA sequences which, if not rectified, will give rise to heritable mutations. Mismatch repair systems can be divided in two broad classes on the basis of their mechanisms of action (for reviews, see Radman and Wagner, 1986; Claverys and Lacks, 1986; Modrich, 1991). The long patch mismatch repair (LPMR) systems act by removing a number of nucleotides (often 1000-3000) from one strand of a DNA double helix in the region of a mispaired or unpaired base (