Induction of JNK and c-Abl signalling by cisplatin and oxaliplatin in mismatch repair-proficient and -deficient cells.
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Loss of DNA mismatch repair has been observed in a variety of human cancers. Recent studies have shown that loss of DNA mismatch repair results in resistance to cisplatin but not oxaliplatin, suggesting that the mismatch repair proteins serve as a detector for cisplatin but not oxaliplatin adducts. To identify the signal transduction pathways with which the detector communicates, we investigated the effect of loss of DNA mismatch repair on activation of known damage-responsive pathways, and recently reported that cisplatin differentially activates c-Jun NH2-terminal kinase (JNK) and c-Abl in repair-proficient vs.-deficient cells. In the current study, we directly compared differential activation of these pathways by cisplatin vs. oxaliplatin. The results confirm that cisplatin activates JNK kinase 5.7 +/- 1.5 (s.d.)-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that the c-Abl response to cisplatin is completely absent in DNA mismatch repair-deficient cells. In contrast, there was no detectable activation of the JNK or c-Abl kinases in DNA mismatch repair-proficient or -deficient cells exposed to oxaliplatin. The present study demonstrates that, despite the similarity of the adducts produced by cisplatin and oxaliplatin, they appear to be recognized by different detectors. The DNA mismatch repair system plays an important part in the recognition of cisplatin adducts, and activation of both the JNK and c-Abl kinases in response to cisplatin damage is dependent on the detector function of the DNA mismatch repair proteins. In contrast, this detector does not respond to oxaliplatin adducts. (+info)
The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?
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Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair. (+info)
CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates.
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We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood. (+info)
The role of the mismatch repair machinery in regulating mitotic and meiotic recombination between diverged sequences in yeast.
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Nonidentical recombination substrates recombine less efficiently than do identical substrates in yeast, and much of this inhibition can be attributed to action of the mismatch repair (MMR) machinery. In this study an intron-based inverted repeat assay system has been used to directly compare the rates of mitotic and meiotic recombination between pairs of 350-bp substrates varying from 82% to 100% in sequence identity. The recombination rate data indicate that sequence divergence impacts mitotic and meiotic recombination similarly, although subtle differences are evident. In addition to assessing recombination rates as a function of sequence divergence, the endpoints of mitotic and meiotic recombination events involving 94%-identical substrates were determined by DNA sequencing. The endpoint analysis indicates that the extent of meiotic heteroduplex DNA formed in a MMR-defective strain is 65% longer than that formed in a wild-type strain. These data are consistent with a model in which the MMR machinery interferes with the formation and/or extension of heteroduplex intermediates during recombination. (+info)
Removal of one nonhomologous DNA end during gene conversion by a RAD1- and MSH2-independent pathway.
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Repair of a double-strand break (DSB) by homologous recombination depends on the invasion of a 3'-ended strand into an intact template sequence to initiate new DNA synthesis. When the end of the invading DNA is not homologous to the donor, the nonhomologous sequences must be removed before new synthesis can begin. In Saccharomyces cerevisiae, the removal of these ends depends on both the nucleotide excision repair endonuclease Rad1p/Rad10p and the mismatch repair proteins Msh2p/Msh3p. In rad1 or msh2 mutants, when both ends of the DSB have nonhomologous ends, repair is reduced approximately 90-fold compared to a plasmid with perfect ends; however, with only one nonhomologous end, repair is reduced on average only 5-fold. These results suggest that yeast has an alternative, but less efficient, way to remove a nonhomologous tail from the second end participating in gene conversion. When the removal of one nonhomologous end is impaired in rad1 and msh2 mutants, there is also a 1-hr delay in the appearance of crossover products of gene conversion, compared to noncrossovers. We interpret these results in terms of the formation and resolution of alternative intermediates of a synthesis-dependent strand annealing mechanism. (+info)
Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth.
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Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomyces cerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol alpha. DNA2 alleles were isolated that cause either temperature sensitivity, sensitivity to alkylation damage, or both. The alkylation-sensitive alleles clustered in the helicase domain, including changes in residues required for helicase activity in related proteins. Additional mutations known or expected to destroy the ATPase and helicase activities of Dna2 were constructed and found to support growth on some media but to cause alkylation sensitivity. Only damage-sensitive alleles were lethal in combination with a ctf4 deletion. Full activity of the Dna2 helicase function is therefore not needed for viability, but is required for repairing damage and for tolerating loss of Ctf4. Arrest of dna2 mutants was RAD9 dependent, but deleting this checkpoint resulted in either no effect or suppression of defects, including the synthetic lethality with ctf4. Dna2 therefore appears to act in repair or lagging strand synthesis together with Pol alpha and Ctf4, in a role that is optimal with, but does not require, full helicase activity. (+info)
Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein.
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We have used immunofluorescent localization to examine the distribution of MLH1 (MutL homolog) foci on synaptonemal complexes (SCs) from juvenile male mice. MLH1 is a mismatch repair protein necessary for meiotic recombination in mice, and MLH1 foci have been proposed to mark crossover sites. We present evidence that the number and distribution of MLH1 foci on SCs closely correspond to the number and distribution of chiasmata on diplotene-metaphase I chromosomes. MLH1 foci were typically excluded from SC in centromeric heterochromatin. For SCs with one MLH1 focus, most foci were located near the middle of long SCs, but near the distal end of short SCs. For SCs with two MLH1 foci, the distribution of foci was bimodal regardless of SC length, with most foci located near the proximal and distal ends. The distribution of MLH1 foci indicated interference between foci. We observed a consistent relative distance (percent of SC length in euchromatin) between two foci on SCs of different lengths, suggesting that positive interference between MLH1 foci is a function of relative SC length. The extended length of pachytene SCs, as compared to more condensed diplotene-metaphase I bivalents, makes mapping crossover events and interference distances using MLH1 foci more accurate than using chiasmata. (+info)
Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA.
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The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila. (+info)