Mismatch Repair Proteins Regulate Heteroduplex Formation during Mitotic Recombination in Yeast

Mismatch repair (MMR) proteins actively inhibit recombination between diverged sequences in both prokaryotes and eukaryotes. Although the molecular basis of the antirecombination activity exerted by MMR proteins is unclear, it presumably involves the recognition of mismatches present in heteroduplex recombination intermediates. This recognition could be exerted during the initial stage of strand exchange, during the extension of heteroduplex DNA, or during the resolution of recombination intermediates. We previously used an assay system based on 350-bp inverted-repeat substrates to demonstrate that MMR proteins strongly inhibit mitotic recombination between diverged sequences in Saccharomyces cerevisiae. The assay system detects only those events that reverse the orientation of the region between the recombination substrates, which can occur as a result of either intrachromatid crossover or sister chromatid conversion. In the present study we sequenced the products of mitotic recombination between 94%-identical substrates in order to map gene conversion tracts in wild-type versus MMR-defective yeast strains. The sequence data indicate that (i) most recombination occurs via sister chromatid conversion and (ii) gene conversion tracts in an MMR-defective strain are significantly longer than those in an isogenic wild-type strain. The shortening of conversion tracts observed in a wild-type strain relative to an MMR-defective strain suggests that at least part of the antirecombination activity of MMR proteins derives from the blockage of heteroduplex extension in the presence of mismatches.     

动物线粒体DNA重组的研究进展

动物线粒体DNA作为遗传标记广泛用于从种内到高级阶元的许多生物学领域,这些应用是建立在线粒体DNA的严格母系遗传方式和不发生重组的基础上的。近年来的研究提出了一些能够证明动物mtDNA发生重组的直接和间接证据。动物mtDNA重组可能主要通过两条途径发生,一条途径是母系mtDNA与核基因组中mtDNA假基因间发生重组;另一条途径是通过父系渗漏引起的不同单倍型的双亲mtDNA间发生重组。父系渗漏是最可能的途径。如果动物界广泛存在线粒体DNA重组,将会对以mtDNA严格母系遗传为基础的许多应用领域产生重要影响。     

Mismatch repair proteins: key regulators of genetic recombination

Mismatch repair (MMR) systems are central to maintaining genome stability in prokaryotes and eukaryotes. MMR proteins play a fundamental role in avoiding mutations, primarily by removing misincorporation errors that occur during DNA replication. MMR proteins also act during genetic recombination in steps that include repairing mismatches in heteroduplex DNA, modulating meiotic crossover control, removing 3' non-homologous tails during double-strand break repair, and preventing recombination between divergent sequences. In this review we will, first, discuss roles for MMR proteins in repairing mismatches that occur during recombination, particularly during meiosis. We will also explore how studying this process has helped to refine models of double-strand break repair, and particularly to our understanding of gene conversion gradients. Second, we will examine the role of MMR proteins in repressing homeologous recombination, i.e. recombination between divergent sequences. We will also compare the requirements for MMR proteins in preventing homeologous recombination to the requirements for these proteins in mismatch repair.     

A novel role for DNA mismatch repair and the autophagic processing of chemotherapy drugs in human tumor cells

DNA Mismatch repair (MMR) maintains genome integrity by correcting DNA replication errors and blocking homologous recombination between divergent DNA sequences. The MMR system also activates both checkpoint and apoptotic responses following certain types of DNA damage. In a recent study, we describe a novel role for MMR in mediating an autophagic response to 6?thioguanine (6-TG), a DNA modifying chemical. Our results show that MMR proteins (MLH1 or MSH2) are required for signaling to the autophagic pathway after exposure to 6-TG. Using PFT-alpha, a p53 inhibitor, and shRNA-mediated silencing of p53 expression, we also show that p53 plays an essential role in the autophagic pathway downstream of the MMR system. This study suggests a novel function of MMR in mediating autophagy following chemical (6-TG) DNA mismatch damage through p53 activation. Here, we present the model and the clinical implications of the role of MMR in autophagy.     

Direct evidence for homologous recombination in mussel (Mytilus galloprovincialis) mitochondrial DNA.

The assumption that animal mitochondrial DNA (mtDNA) does not undergo homologous recombination is based on indirect evidence, yet it has had an important influence on our understanding of mtDNA repair and mutation accumulation (and thus mitochondrial disease and aging) and on biohistorical inferences made from population data. Recently, several studies have suggested recombination in primate mtDNA on the basis of patterns of frequency distribution and linkage associations of mtDNA mutations in human populations, but others have failed to produce similar evidence. Here, we provide direct evidence for homologous mtDNA recombination in mussels, where heteroplasmy is the rule in males. Our results indicate a high rate of mtDNA recombination. Coupled with the observation that mammalian mitochondria contain the enzymes needed for the catalysis of homologous recombination, these findings suggest that animal mtDNA molecules may recombine regularly and that the extent to which this generates new haplotypes may depend only on the frequency of biparental inheritance of the mitochondrial genome. This generalization must, however, await evidence from animal species with typical maternal mtDNA inheritance.     

Role of proliferating cell nuclear antigen interactions in the mismatch repair-dependent processing of mitotic and meiotic recombination intermediates in yeast

The mismatch repair (MMR) system is critical not only for the repair of DNA replication errors, but also for the regulation of mitotic and meiotic recombination processes. In a manner analogous to its ability to remove replication errors, the MMR system can remove mismatches in heteroduplex recombination intermediates to generate gene conversion events. Alternatively, such mismatches can trigger an MMR-dependent antirecombination activity that blocks the completion of recombination, thereby limiting interactions between diverged sequences. In Saccharomyces cerevisiae, the MMR proteins Msh3, Msh6, and Mlh1 interact with proliferating cell nuclear antigen (PCNA), and mutations that disrupt these interactions result in a mutator phenotype. In addition, some mutations in the PCNA-encoding POL30 gene increase mutation rates in an MMR-dependent manner. In the current study, pol30, mlh1, and msh6 mutants were used to examine whether MMR-PCNA interactions are similarly important during mitotic and meiotic recombination. We find that MMR-PCNA interactions are important for repairing mismatches formed during meiotic recombination, but play only a relatively minor role in regulating the fidelity of mitotic recombination.     

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.     

Mismatch repair proteins and mitotic genome stability

Mismatch repair (MMR) proteins play a critical role in maintaining the mitotic stability of eukaryotic genomes. MMR proteins repair errors made during DNA replication and in their absence, mutations accumulate at elevated rates. In addition, MMR proteins inhibit recombination between non-identical DNA sequences, and hence prevent genome rearrangements resulting from interactions between repetitive elements. This review provides an overview of the anti-mutator and anti-recombination functions of MMR proteins in the yeast Saccharomyces cerevisiae.     

Evidence for the lack of mismatch-repair directed antirecombination during mouse meiosis

Meiotic recombination was studied in DNA mismatch repair (MMR)-deficient mice using a strain carrying a Pms2 knockout mutation. Using single-sperm typing, recombination was analyzed over five intervals on four chromosomes in four Pms2 -/- animals. A total of 1936 meioses were studied and compared to 1848 meioses from three Pms2 +/+ controls. A smaller study was carried out on a single interval in each of two chromosomes in an MMR-deficient mouse homozygous for the Msh2 knockout mutation. A total of 792 meioses were examined in the Msh2 -/- and 880 meioses in the Msh2 +/+ animal. Recombination fractions were not significantly different in either of the MMR-deficient mouse strains when compared to MMR-proficient controls. Our results appear to conflict with mouse embryonic stem (ES) cell gene-targeting experiments where MMR plays a major role in determining the efficiency of homologous recombination between nonidentical sequences. A number of possibilities could explain the apparent lack of a significant effect on meiosis.     

A reevaluation of rice mitochondrial evolution based on the complete sequence of male-fertile and male-sterile mitochondrial genomes

Plant mitochondrial genomes have features that distinguish them radically from their animal counterparts: a high rate of rearrangement, of uptake and loss of DNA sequences, and an extremely low point mutation rate. Perhaps the most unique structural feature of plant mitochondrial DNAs is the presence of large repeated sequences involved in intramolecular and intermolecular recombination. In addition, rare recombination events can occur across shorter repeats, creating rearrangements that result in aberrant phenotypes, including pollen abortion, which is known as cytoplasmic male sterility (CMS). Using next-generation sequencing, we pyrosequenced two rice (Oryza sativa) mitochondrial genomes that belong to the indica subspecies. One genome is normal, while the other carries the wild abortive-CMS. We find that numerous rearrangements in the rice mitochondrial genome occur even between close cytotypes during rice evolution. Unlike maize (Zea mays), a closely related species also belonging to the grass family, integration of plastid sequences did not play a role in the sequence divergence between rice cytotypes. This study also uncovered an excellent candidate for the wild abortive-CMS-encoding gene; like most of the CMS-associated open reading frames that are known in other species, this candidate was created via a rearrangement, is chimeric in structure, possesses predicted transmembrane domains, and coopted the promoter of a genuine mitochondrial gene. Our data give new insights into rice mitochondrial evolution, correcting previous reports.     

How clonal are human mitochondria?

Phylogenetic trees constructed using human mitochondrial sequences contain a large number of homoplasies. These are due either to repeated mutation or to recombination between mitochondrial lineages. We show that a tree constructed using synonymous variation in the protein coding sequences of 29 largely complete human mitochondrial molecules contains 22 homoplasies at 32 phylogenetically informative sites. This level of homoplasy is very unlikely if inheritance is clonal, even if we take into account base composition bias. There must either be 'hypervariable' sites or recombination between mitochondria. We present evidence which suggests that hypervariable sites do not exist in our data. It therefore seems likely that recombination has occurred between mitochondrial lineages in humans.     

Role of mismatch repair in the fidelity of RAD51- and RAD59-dependent recombination in Saccharomyces cerevisiae

To prevent genome instability, recombination between sequences that contain mismatches (homeologous recombination) is suppressed by the mismatch repair (MMR) pathway. To understand the interactions necessary for this regulation, the genetic requirements for the inhibition of homeologous recombination were examined using mutants in the RAD52 epistasis group of Saccharomyces cerevisiae. The use of a chromosomal inverted-repeat recombination assay to measure spontaneous recombination between 91 and 100% identical sequences demonstrated differences in the fidelity of recombination in pathways defined by their dependence on RAD51 and RAD59. In addition, the regulation of homeologous recombination in rad51 and rad59 mutants displayed distinct patterns of inhibition by different members of the MMR pathway. Whereas the requirements for the MutS homolog, MSH2, and the MutL homolog, MLH1, in the suppression of homeologous recombination were similar in rad51 strains, the loss of MSH2 caused a greater loss in homeologous recombination suppression than did the loss of MLH1 in a rad59 strain. The nonequivalence of the regulatory patterns in the wild-type and mutant strains suggests an overlap between the roles of the RAD51 and RAD59 gene products in potential cooperative recombination mechanisms used in wild-type cells.     

Distinct roles for the Saccharomyces cerevisiae mismatch repair proteins in heteroduplex rejection, mismatch repair and nonhomologous tail removal

The Saccharomyces cerevisiae mismatch repair (MMR) protein MSH6 and the SGS1 helicase were recently shown to play similarly important roles in preventing recombination between divergent DNA sequences in a single-strand annealing (SSA) assay. In contrast, MMR factors such as Mlh1p, Pms1p, and Exo1p were shown to not be required or to play only minimal roles. In this study we tested mutations that disrupt Sgs1p helicase activity, Msh2p-Msh6p mismatch recognition, and ATP binding and hydrolysis activities for their effect on preventing recombination between divergent DNA sequences (heteroduplex rejection) during SSA. The results support a model in which the Msh proteins act with Sgs1p to unwind DNA recombination intermediates containing mismatches. Importantly, msh2 mutants that displayed separation-of-function phenotypes with respect to nonhomologous tail removal during SSA and heteroduplex rejection were characterized. These studies suggest that nonhomologous tail removal is a separate function of Msh proteins that is likely to involve a distinct DNA binding activity. The involvement of Sgs1p in heteroduplex rejection but not nonhomologous tail removal further illustrates that subsets of MMR proteins collaborate with factors in different DNA repair pathways to maintain genome stability.     

A Novel Role for DNA Mismatch Repair and the Autophagic Processing of Chemotherapy Drugs in Human Tumor Cells

DNA Mismatch repair (MMR) maintains genome integrity by correcting DNA replication errors and blocking homologous recombination between divergent DNA sequences. The MMR system also activates both checkpoint and apoptotic responses following certain types of DNA damage. In a recent study, we describe a novel role for MMR in mediating an autophagic response to 6-thioguanine (6-TG), a DNA modifying chemical. Our results show that MMR proteins (MLH1 or MSH2) are required for signaling to the autophagic pathway after exposure to 6-TG. Using PFT-α, a p53 inhibitor, and shRNA-mediated silencing of p53 expression, we also show that p53 plays an essential role in the autophagic pathway downstream of the MMR system. This study suggests a novel function of MMR in mediating autophagy following chemical (6-TG) DNA mismatch damage through p53 activation. Here, we present the model and the clinical implications of the role of MMR in autophagy.

Addendum to:

DNA Mismatch Repair Initiates 6-Thioguanine-Induced Autophagy through p53 Activation in Human Tumor Cells

X. Zeng, T. Yan, J.E. Schupp, Y. Seo and T.J. Kinsella

Clin Cancer Res 2007; 13:1315-21     

Animal mitochondrial DNA as a genetic marker in population and evolutionary biology

Animal mitochondrial DNA (mtDNA) is playing an increasingly important role as a genetic marker in population and evolutionary biology. The popularity of this molecule derives, in part, from the relative ease with which clearly homologous sequences can be isolated and compared. Simple sequence organization, maternal inheritance and absence of recombination make mtDNA an ideal marker for tracing maternal genealogies. Rapid rate of sequence divergence (at least in vertebrates) allows discrimination of recently diverged lineages. Studies of mtDNAs from a diversity of animal groups have revealed significant variation among taxa in mtDNA sequence dynamics, gene order and genome size. They have also provided important insights into population structure, geographic variation, zoogeography and phylogeny.     

Recombination sequences in plant mitochondrial genomes: diversity and homologies to known mitochondrial genes.

Several plant mitochondrial genomes contain repeated sequences that are postulated to be sites of homologous intragenomic recombination (1-3). In this report, we have used filter hybridizations to investigate sequence relationships between the cloned mitochondrial DNA (mtDNA) recombination repeats from turnip, spinach and maize and total mtDNA isolated from thirteen species of angiosperms. We find that strong sequence homologies exist between the spinach and turnip recombination repeats and essentially all other mitochondrial genomes tested, whereas a major maize recombination repeat does not hybridize to any other mtDNA. The sequences homologous to the turnip repeat do not appear to function in recombination in any other genome, whereas the spinach repeat hybridizes to reiterated sequences within the mitochondrial genomes of wheat and two species of pokeweed that do appear to be sites of recombination. Thus, although intragenomic recombination is a widespread phenomenon in plant mitochondria, it appears that different sequences either serve as substrates for this function in different species, or else surround a relatively short common recombination site which does not cross-hybridize under our experimental conditions. Identified gene sequences from maize mtDNA were used in heterologous hybridizations to show that the repeated sequences implicated in recombination in turnip and spinach/pokeweed/wheat mitochondria include, or are closely linked to genes for subunit II of cytochrome c oxidase and 26S rRNA, respectively. Together with previous studies indicating that the 18S rRNA gene in wheat mtDNA is contained within a recombination repeat (3), these results imply an unexpectedly frequent association between recombination repeats and plant mitochondrial genes.     

"Patchy-tachy" leads to false positives for recombination

Indirect tests have detected recombination in mitochondrial DNA (mtDNA) from many animal lineages, including mammals. However, it is possible that features of the molecular evolutionary process without recombination could be incorrectly inferred by indirect tests as being due to recombination. We have identified one such example, which we call "patchy-tachy" (PT), where different partitions of sequences evolve at different rates, that leads to an excess of false positives for recombination inferred by indirect tests. To explore this phenomena, we characterized the false positive rates of six widely used indirect tests for recombination using simulations of general models for mtDNA evolution with PT but without recombination. All tests produced 30-99% false positives for recombination, although the conditions that produced the maximal level of false positives differed between the tests. To evaluate the degree to which conditions that exacerbate false positives are found in published sequence data, we turned to 20 animal mtDNA data sets in which recombination is suggested by indirect tests. Using a model where different regions of the sequences were free to evolve at different rates in different lineages, we demonstrated that PT is prevalent in many data sets in which recombination was previously inferred using indirect tests. Taken together, our results argue that PT without recombination is a viable alternative explanation for detection of widespread recombination in animal mtDNA using indirect tests.     

Examination of the roles of Sgs1 and Srs2 helicases in the enforcement of recombination fidelity in Saccharomyces cerevisiae

Mutation in SGS1, which encodes the yeast homolog of the human Bloom helicase, or in mismatch repair (MMR) genes confers defects in the suppression of mitotic recombination between similar but nonidentical (homeologous) sequences. Mutational analysis of SGS1 suggests that the helicase activity is required for the suppression of both homologous and homeologous recombination and that the C-terminal 200 amino acids may be required specifically for the suppression of homeologous recombination. To clarify the mechanism by which the Sgs1 helicase enforces the fidelity of recombination, we examined the phenotypes associated with SGS1 deletion in MMR-defective and recombination-defective backgrounds. Deletion of SGS1 caused no additional loss of recombination fidelity above that associated with MMR defects, indicating that the suppression of homeologous recombination by Sgs1 may be dependent on MMR. However, the phenotype of the sgs1 rad51 mutant suggests a MMR-independent role of Sgs1 in the suppression of RAD51-independent recombination. While homologous recombination levels increase in sgs1Delta and in srs2Delta strains, the suppression of homeologous recombination was not relaxed in the srs2 mutant. Thus, although both Sgs1 and Srs2 limit the overall level of mitotic recombination, there are distinct differences in the roles of these helicases with respect to enforcement of recombination fidelity.     

Effects of suppressing the DNA mismatch repair system on homeologous recombination in tomato

In plant breeding, the ability to manipulate genetic (meiotic) recombination would be beneficial for facilitating gene transfer from wild relatives of crop plants. The DNA mismatch repair (MMR) system helps maintain genetic integrity by correcting base mismatches that arise via DNA synthesis or damage, and antagonizes recombination between homeologous (divergent) DNA sequences. Previous studies have established that the genomes of cultivated tomato (Solanum lycopersicum) and the wild relative S. lycopersicoides are substantially diverged (homeologous) such that recombination between their chromosomes is strongly reduced. Here, we report the effects on homeologous recombination of suppressing endogenous MMR genes in S. lycopersicum via RNAi-induced silencing of SlMSH2 and SlMSH7 or overexpressing dominant negatives of Arabidopsis MSH2 (AtMSH2-DN) in an alien substitution line (SL-8) of S. lycopersicoides in tomato. We show that certain inhibitions of MMR (RNAi of SlMSH7, AtMSH2-DN) are associated with modest increases in homeologous recombination, ranging from 3.8 to 29.2% (average rate of 17.8%) compared to controls. Unexpectedly, only the AtMSH2-DN proteins but not RNAi-induced silencing of MSH2 was found to increase homeologous recombination. The ratio of single to double crossovers (SCO:DCO ratio) decreased by approximately 50% in progeny of the AtMSH2-DN parents. An increase in the frequency of heterozygous SL-8 plants was also observed in the progeny of the SlMSH7-RNAi parents. Our findings may contribute to acceleration of introgression in cultivated tomato.