Loss of DNA mismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin.
(33/1695)
In addition to recognizing and repairing mismatched bases in DNA, the mismatch repair (MMR) system also detects cisplatin DNA adducts and loss of MMR results in resistance to cisplatin. A comparison was made of the ability of MMR-proficient and -deficient cells to remove cisplatin adducts from their genome and to reactivate a transiently transfected plasmid that had previously been inactivated by cisplatin to express the firefly luciferase enzyme. MMR deficiency due to loss of hMLH1 function did not change the extent of platinum (Pt) accumulation or kinetics of removal from total cellular DNA. However, MMR-deficient cells, lacking either hMLH1 or hMSH2, generated twofold more luciferase activity from a cisplatin-damaged reporter plasmid than their MMR-proficient counterparts. Thus, detection of the cisplatin adducts by the MMR system reduced the efficiency of reactivation of the damaged luciferase gene compared to cells lacking this detector. The twofold reduction in reactivation efficiency was of the same order of magnitude as the difference in cisplatin sensitivity between the MMR-proficient and -deficient cells. We conclude that although MMR-proficient and -deficient cells remove Pt from their genome at equal rates, the loss of a functional MMR system facilitates the reactivation of a cisplatin-damaged reporter gene. (+info)
Correction of large mispaired DNA loops by extracts of Saccharomyces cerevisiae.
(34/1695)
Single base mispairs and small loops are corrected by DNA mismatch repair, but little is known about the correction of large loops. In this paper, large loop repair was examined in nuclear extracts of yeast. Biochemical assays showed that repair activity occurred on loops of 16, 27, and 216 bases, whereas a G-T mispair and an 8-base loop were poorly corrected under these conditions. Two modes of loop repair were revealed by comparison of heteroduplexes that contained a site-specific nick or were covalently closed. A nick-stimulated repair mode directs correction to the discontinuous strand, regardless of which strand contains the loop. An alternative mode is nick-independent and preferentially removes the loop. Both outcomes of repair were largely eliminated when DNA replication was inhibited, suggesting a requirement for repair synthesis. Excision tracts of 100-200 nucleotides, spanning the position of the loop, were observed on each strand under conditions of limited DNA repair synthesis. Both repair modes were independent of the mismatch correction genes MSH2, MSH3, MLH1, and PMS1, as judged by activity in mutant extracts. Together the loop specificity and mutant results furnish evidence for a large loop repair pathway in yeast that is distinct from mismatch repair. (+info)
Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation.
(35/1695)
During maturation of the immune response, IgM+ B cells switch to expression of one of the downstream isotypes (IgG, A or E). This class switching occurs by region-specific recombination within the IgH locus through an unknown mechanism. A lack of switch recombination in mice deficient in components of the DNA-dependent protein kinase (DNA-PK)-Ku complex has pointed to a role for non-homologous end joining. Here we characterize a switching defect in mice lacking a protein involved in DNA mismatch recognition. Mice deficient in Msh2 give diminished IgG (but not IgM) responses following challenge with both T cell-dependent and T cell-independent antigens. This appears to reflect a B cell-intrinsic defect since B cells from Msh2-deficient mice also exhibit impaired switching (but not blasting or proliferation) on in vitro culture with lipopolysaccharide. Furthermore, those switches that do occur in Msh2-deficient B cells reveal a shift in the distribution of recombination sites used: the breakpoints are more likely to occur in consensus motifs. These results, which intriguingly parallel the effects of Msh2 deficiency on hypermutation, suggest a role for Msh2 in the mechanics of class-switch recombination. (+info)
A Uve1p-mediated mismatch repair pathway in Schizosaccharomyces pombe.
(36/1695)
UV damage endonuclease (Uve1p) from Schizosaccharomyces pombe was initially described as a DNA repair enzyme specific for the repair of UV light-induced photoproducts and proposed as the initial step in an alternative excision repair pathway. Here we present biochemical and genetic evidence demonstrating that Uve1p is also a mismatch repair endonuclease which recognizes and cleaves DNA 5' to the mispaired base in a strand-specific manner. The biochemical properties of the Uve1p-mediated mismatch endonuclease activity are similar to those of the Uve1p-mediated UV photoproduct endonuclease. Mutants lacking Uve1p display a spontaneous mutator phenotype, further confirming the notion that Uve1p plays a role in mismatch repair. These results suggest that Uve1p has a surprisingly broad substrate specificity and may function as a general type of DNA repair protein with the capacity to initiate mismatch repair in certain organisms. (+info)
Removal of frameshift intermediates by mismatch repair proteins in Saccharomyces cerevisiae.
(37/1695)
Frameshift mutations occur when the coding region of a gene is altered by addition or deletion of a number of base pairs that is not a multiple of three. The occurrence of a deletion versus an insertion type of frameshift depends on the nature of the transient intermediate structure formed during DNA synthesis. Extrahelical bases on the template strand give rise to deletions, whereas extrahelical bases on the strand being synthesized produce insertions. We previously used reversion of a +1 frameshift mutation to analyze the role of the mismatch repair (MMR) machinery in correcting -1 frameshift intermediates within a defined region of the yeast LYS2 gene. In this study, we have used reversion of a -1 frameshift mutation within the same region of LYS2 to analyze the role of the MMR machinery in the correction of frameshift intermediates that give rise to insertion events. We found that insertion and deletion events occur at similar rates but that the reversion spectra are very different in both the wild-type and MMR-defective backgrounds. In addition, analysis of the +1 spectra revealed novel roles for Msh3p and Msh6p in removing specific types of frameshift intermediates. (+info)
Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments.
(38/1695)
Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these hands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 microM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 microM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 microM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo. (+info)
Molecular diagnostics of cancer predisposition: hereditary non-polyposis colorectal carcinoma and mismatch repair defects.
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Hereditary non-polyposis colorectal carcinoma accounts for 5-13% of all colorectal carcinomas and is inherited in a dominant fashion. Two different forms can be distinguished. Type I is restricted to colorectal cancers, whereas type II patients acquire acolorectal, endometrial, gastric, small intestinal and transitional carcinomas of the upper urinary tract. Germline mutations in the human mismatch repair genes (hMSH2, hMSH6, hMLH1, hPMS2) account for the majority of hereditary non-polyposis colorectal carcinoma. As a result of the mismatch repair deficiency, replication errors are not repaired, resulting in a mutator phenotype. Simple repetitive sequences (microsatellites) are especially prone to replication errors and analysis of their stability combined with immunohistochemical analysis of mismatch repair protein expression provides a rapid diagnostic strategy. For patients either (1) fulfilling the Amsterdam criteria for HNPCC, (2) with synchronous or metachronous hereditary non-polyposis colorectal carcinoma-related tumors, (3) with hereditary non-polyposis colorectal carcinoma-related tumors before the age of 45 and/or (4) with right sided CRC and mucinous, solid, or cribriform growth patterns, screening for mismatch repair deficiencies should be performed. The identification of colorectal cancers displaying a mutator phenotype has implications for both treatment and prognosis. (+info)
Sequence-dependent mutations in a shuttle vector plasmid replicated in a mismatch repair deficient human cell line.
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We utilized a shuttle vector plasmid (pLSC) to assess the role of DNA sequence and mismatch repair on mutagenesis in human cells. pLSC contains an interrupted 29 bp mononucleotide poly(G) run within a bacterial suppressor tRNA gene, which acts as a highly sensitive mutagenic target for detection of base substitution and frameshift mutations. The frequency of spontaneous mutations in pLSC was found to be similar after replication in either the hMSH6 (GT binding protein) mismatch repair-deficient MT1 line or its parental, mismatch repair-proficient line, TK6. However, the classes of plasmid mutations showed distinct differences in the two cell lines. Single base deletions comprised 48% of the mutations in the 56 independent pLSC plasmids sequenced from MT1 cells while these represented only 18% of the 40 independent pLSC mutants sequenced from the wild-type TK6 cells (P = 0.001). Virtually all the deletions included the mononucleotide run. In contrast, in pSP189, which contains the unmodified supF tRNA without the mononucleotide sequence, no single base deletions were observed for either cell line (P < 0.001). UV treatment of pLSC and pSP189 resulted in a 12-140-fold increase in mutations in TK6 and MT1 cells. These were predominately single base substitution mutations without a large increase in deletion mutations in the mononucleotide run in pLSC. These data indicate that a mononucleotide poly(G) run promotes single base deletion mutations. This effect is enhanced in a hMSH6 mismatch repair-deficient cell line and is independent of UV-induced mutagenesis. (+info)