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Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.

In yeast, DNA breaks are usually repaired by homologous recombination (HR). An early step for HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, there will be mismatches within the heteroduplex DNA (hetDNA). In wild-type strains, these mismatches are repaired by the mismatch repair (MMR) system, producing a gene conversion event. In strains lacking MMR, the mismatches persist. Most previous studies involving hetDNA formed during mitotic recombination were restricted to one locus. Below, we present a global mapping of hetDNA formed in the MMR-defective mlh1 strain. We find that many recombination events are associated with repair of double-stranded DNA gaps and/or involve Mlh1-independent mismatch repair. Many of our events are not explicable by the simplest form of the double-strand break repair model of recombination.

Homologous recombination (HR), the most significant event in meiosis, has important implications for genetic diversity and evolution in organisms. Heteroduplex DNA (hDNA), the product of HR, can be captured by artificially induced chromosome doubling during the development of the embryo sac to inhibit postmeiotic segregation, subsequently, and hDNAs are directly detected using codominant simple sequence repeat (SSR) markers. In the present study, two hybrid triploid populations derived from doubling the chromosomes of the embryo sac induced by high temperature in Populus tomentosa served as starting materials. Eighty-seven, 62, and 79 SSR markers on chromosomes 01, 04, and 19, respectively, that were heterozygous in the maternal parent and different from the paternal parent were screened to detect and characterize the hDNA in P. tomentosa. The results showed that the hDNA frequency patterns on chromosomes changed slightly when the number of SSR primers increased. The highest hDNA frequency occurred at the adjacent terminal on chromosomes, which was slightly higher than those at the terminals in the two genotypic individuals, and the hDNA frequency gradually decreased as the locus-centromere distance decreased. With the increase in the number of SSR markers employed for detection, the number of recombination events (REs) detected significantly increased. In regions with high methylation or long terminal repeat (LTR) retrotransposon enrichment, the frequency of hDNA was low, and high frequencies were observed in regions with low sequence complexity and high gene density. High-frequency recombination occurring at high gene density regions strongly affected the association between molecular markers and quantitative trait loci (QTLs), which was an important factor contributing to the difficulty encountered by MAS in achieving the expected breeding results.

Homologous recombination (HR), the most significant event in meiosis, has important implications for genetic diversity and evolution in organisms. Heteroduplex DNA (hDNA), the product of HR, can be captured by artificially induced chromosome doubling during the development of the embryo sac to inhibit postmeiotic segregation, subsequently, and hDNAs are directly detected using codominant simple sequence repeat (SSR) markers. In the present study, two hybrid triploid populations derived from doubling the chromosomes of the embryo sac induced by high temperature in Populus tomentosa served as starting materials. Eighty-seven, 62, and 79 SSR markers on chromosomes 01, 04, and 19, respectively, that were heterozygous in the maternal parent and different from the paternal parent were screened to detect and characterize the hDNA in P. tomentosa. The results showed that the hDNA frequency patterns on chromosomes changed slightly when the number of SSR primers increased. The highest hDNA frequency occurred at the adjacent terminal on chromosomes, which was slightly higher than those at the terminals in the two genotypic individuals, and the hDNA frequency gradually decreased as the locus-centromere distance decreased. With the increase in the number of SSR markers employed for detection, the number of recombination events (REs) detected significantly increased. In regions with high methylation or long terminal repeat (LTR) retrotransposon enrichment, the frequency of hDNA was low, and high frequencies were observed in regions with low sequence complexity and high gene density. High-frequency recombination occurring at high gene density regions strongly affected the association between molecular markers and quantitative trait loci (QTLs), which was an important factor contributing to the difficulty encountered by MAS in achieving the expected breeding results. [ABSTRACT FROM AUTHOR]

Eukaryotic genomes contain many duplicated genes closely located with each other, such as the hexose transporter (HXT) genes in Saccharomyces cerevisiae. They can potentially recombine via single-strand annealing (SSA) pathway. SSA between highly divergent sequences generates heteroduplex DNA intermediates with many mismatches, which can be corrected by mismatch repair (MMR), resulting in recombinant sequences with a single junction point. In this report, we demonstrate that SSA between HXT1 and HXT4 genes in MMR-deficient yeast cells produces recombinant genes with multiple-junctions resulting from alternating HXT1 and HXT4 tracts. The mutations in MMR genes had differential effects on SSA frequencies; msh6Δ mutation significantly stimulated SSA events, whereas msh2Δ and msh3Δ slightly suppressed it. We set up an assay that can identify a pair of recombinant genes derived from a single heteroduplex DNA. As a result, the recombinant genes with multiple-junctions were found to accompany genes with single-junctions. Based on the results presented here, a model was proposed to generate multiple-junctions in SSA pathway involving an alternative short-patch repair system. [ABSTRACT FROM AUTHOR]

In yeast, DNA breaks are usually repaired by homologous recombination (HR). An early step for HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, there will be mismatches within the heteroduplex DNA (hetDNA). In wild-type strains, these mismatches are repaired by the mismatch repair (MMR) system, producing a gene conversion event. In strains lacking MMR, the mismatches persist. Most previous studies involving hetDNA formed during mitotic recombination were restricted to one locus. Below, we present a global mapping of hetDNA formed in the MMR-defective mlh1 strain. We find that many recombination events are associated with repair of double-stranded DNA gaps and/or involve Mlh1-independent mismatch repair. Many of our events are not explicable by the simplest form of the double-strand break repair model of recombination.

RAGs (recombination activating genes) are responsible for the generation of antigen receptor diversity through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. In addition to its physiological property, wherein RAG functions as a sequence-specific nuclease, it can also act as a structure-specific nuclease leading to genomic instability and cancer. In the present study, we investigate the factors that regulate RAG cleavage on non-B DNA structures. We find that RAG binding and cleavage on heteroduplex DNA is dependent on the length of the double-stranded flanking region. Besides, the immediate flanking double-stranded region regulates RAG activity in a sequence-dependent manner. Interestingly, the cleavage efficiency of RAGs at the heteroduplex region is influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights on the regulation of the pathological functions of RAGs. [ABSTRACT FROM AUTHOR]

Heteroduplex DNA ( hDNA) generated during homologous recombination (HR) is an important component that shapes genetic diversity in sexually reproducing organisms. However, studies of this process in higher plants are limited. This is because hDNAs are difficult to capture in higher plants as their reproductive developmental model only produces normal gametes and does not preserve the mitotic products of the post-meiotic segregation (PMS) process which is crucial for studying hDNAs. In this study, using the model system for tree and woody perennial plant biology ( Populus), we propose a strategy for characterizing hDNAs in higher plants. We captured hDNAs by constructing triploid hybrids originating from a cross between unreduced 2 n eggs (containing hDNA information as a result of inhibition chromosome segregation at the PMS stage) with normal male gametes. These triploid hybrids allowed us to detect the frequency and location of persistent hDNAs resulting from HR at the molecular level. We found that the frequency of persistent hDNAs, which ranged from 5.3 to 76.6%, was related to locations of the simple sequence repeat markers at the chromosomes, such as the locus-centromere distance, the surrounding DNA sequence and epigenetic information, and the richness of protein-coding transcripts at these loci. In summary, this study provides a method for characterizing persistent hDNAs in higher plants. When high-throughput sequencing techniques can be incorporated, genome-wide persistent hDNA assays for higher plants can be easily carried out using the strategy presented in this study. [ABSTRACT FROM AUTHOR]

It is generally agreed that the protease inhibitor (PI) alleles PI*S (Val264Glu) and PI*Z (Lys342Glu) are the most common alpha 1 antitrypsin deficiency variants worldwide, but the PI*Mmalton allele (ΔPhe52) prevails over these variants in some Mediterranean regions. In eastern Tunisia (Mahdia), we screened 100 subjects with chronic obstructive pulmonary disease for these variants. The PI*S and PI*Z alleles were genotyped by the previously described SexAI/ Hpγ99I RFLP-PCR. We provide here a new method for PI*Mmalton genotyping using mismatched RFLP-PCR. These methods are suitable for routine clinical application and can easily be reproduced by several laboratories, since they do not require extensive optimization, unlike the previously described bidirectional allele-specific amplification PCR for PI*Mmalton genotyping. Our results were in agreement with previous reports from central Tunisia (Kairouan), suggesting that the PI*Mmalton mutation is the most frequent alpha 1 antitrypsin deficiency-related mutation in Tunisia. [ABSTRACT FROM AUTHOR]

Abstract: Mismatch repair (MMR) system maintains genome integrity by correcting mispaired or unpaired bases which have escaped the proofreading activity of DNA polymerases. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in the mechanism vary from prokaryotes to eukaryotes and even between humans and plants. Cells deficient in MMR genes have been observed to display a mutator phenotype characterized by an increased rate in spontaneous mutation, instability of microsatellite sequences and illegitimate recombination between diverged DNA sequences. Studies of the mutator phenotype have demonstrated a critical role for the MMR system in mutation avoidance and genetic stability. Here, we briefly review our current knowledge of the MMR mechanism and then focus on the in vivo biochemical and genetic assays used to investigate the function of the MMR proteins in processing DNA mismatches generated during replication and mitotic recombination in Escherichia coli, Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana. An overview of the biochemical assays developed to study mismatch correction in vitro is also provided. [Copyright &y& Elsevier]

Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3β-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5–21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project. [ABSTRACT FROM AUTHOR]

Abstract: Reassociating double-stranded DNA from single-stranded components is necessary for many molecular genetics experiments. The choice of a DNA reassociation method is dictated by the complexity of the starting material. Reassociation of simple oligomers needs only slow cooling in an aqueous environment, whereas reannealing the many single-stranded DNAs of complex genomic mixtures requires both a phenol emulsion to accelerate DNA reassociation and dedicated equipment to maintain the emulsion. We present a method that is equally suitable for reassociating either simple or complex DNA mixtures. The Oscillating Phenol Emulsion Reassociation Technique (OsPERT) was primarily developed to prepare heteroduplex DNA from alkali-denatured high molecular weight human genomic DNA samples in which hundreds of thousands of fragments need to be reannealed, but the simplicity of the technique makes it practical for less demanding DNA reassociation applications. [Copyright &y& Elsevier]

Abstract: This investigation demonstrates DNA mismatch repair activity in Haemophilus influenzae cell free extracts. The mutS gene as well as purified protein of H. influenzae restored repair activity in complementation assays performed with mutS deficient Escherichia coli strain. The difference in affinity for GT and AC mismatched bases by H. influenzae MutS was reflected in the efficiency with which these DNA heteroduplexes were repaired in vitro, with GT being repaired well and AC the least. Unlike E. coli MutS, the H. influenzae homolog failed to give protein–DNA complex with homoduplex DNA. Interestingly, MutS was found to bind single-stranded DNA but with lesser affinity as compared to heteroduplex DNA. Apart from the nucleotide- and DNA-mediated conformational transitions, as monitored by circular dichroism and limited proteolysis, our data suggest a functional role when H. influenzae MutS encounters single-stranded DNA during exonucleolytic step of DNA repair process. We propose that, conformational changes in H. influenzae MutS not only modulate mismatch recognition but also trigger some of the down stream processes involved in the DNA mismatch repair process. [Copyright &y& Elsevier]

Single- and multi-base (loop) mismatches can arise in DNA by replication errors, during recombination, and by chemical modification of DNA. Single-base and loop mismatches of several nucleotides are efficiently repaired in mammalian cells by a nick-directed, MSH2-dependent mechanism. Larger loop mismatches (≥12 bases) are repaired by an MSH2-independent mechanism. Prior studies have shown that 12- and 14-base palindromic loops are repaired with bias toward loop retention, and that repair bias is eliminated when five single-base mismatches flank the loop mismatch. Here we show that one single-base mismatch near a 12-base palindromic loop is sufficient to eliminate loop repair bias in wild-type, but not MSH2-defective mammalian cells. We also show that palindromic loop and single-base mismatches separated by 12 bases are repaired independently at least 10% of the time in wild-type cells, and at least 30% of the time in MSH2-defective cells. Palindromic loop and single-base mismatches separated by two bases were never repaired independently. These and other data indicate that loop repair tracts are variable in length. All tracts extend at least 2 bases, some extend <12 bases, and others >12 bases, on one side of the loop. These properties distinguish palindromic loop mismatch repair from the three known excision repair pathways: base excision repair which has one to six base tracts, nucleotide excision repair which has ∼30 base tracts, and MSH2-dependent mismatch repair, which has tracts that extend for several hundred bases. [Copyright &y& Elsevier]

Functional deficiency of mismatch repair (MMR) system is one of the mechanisms of tumorigenesis. With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZα gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G·G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (mutS-) without the MMR ability on the indicator plate with x-gal and IPTG, there are three kinds of plaques, mixture plaque as the characteristic phenotype of heteroduplex DNA, blue and clear plaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100x (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functional status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to GG is more than 50%. The Lovo efficiency to del(2) is less than 10%, to GC is less than 20 %. Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target, TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (microsatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the function status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors. [ABSTRACT FROM AUTHOR]

Antimutators are genetic mutants that produce mutations at reduced rates compared to the wild type strain. They are interesting because they may provide insights into the mechanisms by which spontaneous mutations occur. We have investigated a reported antimutator strain of Escherichia coli termed mud for its possible mechanism. The mud strain exhibits a decrease in both spontaneous mutagenesis and mutability with alkylated agents and base analogs. These types of DNA lesions are known to be the substrates for the E. coli methyl-directed mismatch repair encoded by the mutHLSU system. We investigated whether the putative antimutator effect results from the increased expression or activity of the mutHLSU system. To directly measure the mismatch repair capacity of mud cells, we have transfected them with phage λ heteroduplexes and scored the fraction of mixed (unrepaired) infective centers. This transfection system has been used routinely to assay mismatch repair capacity in E. coli and other organisms. No difference between mud and wild type cells is observed. From the results of the experiments we conclude that the reported antimutator effect of mud does not result from enhanced mismatch repair capacity. This conclusion is consistent with recently published evidence that the mud effect does not represent a real antimutator effect, but is an artifact due to impaired growth of mud cells under certain selective conditions. [Copyright &y& Elsevier]

The Arabidopsis thaliana MSH2 (AtMSH2) gene encodes a protein that belongs to a family of highly conserved proteins (MutS homologues (MSH)) involved in DNA mismatch repair. Sequence analysis strongly suggests that this single copy gene is indeed a homologue of MSH2, a gene known to play a central role in eukaryotic mismatch repair. In this report, we show that the AtMSH2 protein has functional attributes characteristic of previously described mismatch repair proteins. First, over-expression of this protein in Escherichia coli leads to a mutator phenotype similar to that reported previously for known functional homologues. Second, gel retardation assays revealed that the AtMSH2 protein has a 10-fold greater affinity for DNA containing a single pair of mismatched nucleotides versus perfectly matched DNA. These results provide experimental evidence that AtMSH2 is indeed a functional homologue of MutS.Key words: DNA mismatch repair, heteroduplex DNA, mutation rate.Le gène MSH2 d'Arabidopsis thaliana (AtMSH2) code pour une protéine qui appartient à une famille de protéines très conservées (MutS homologues (MSH)) lesquelles participent à la correction des mésappariements au sein de l'ADN. L'examen de la séquence de ce gène à copie unique suggère fortement que celui-ci est véritablement un homologue de MSH2, un des gènes eucaryotiques jouant un rôle central dans la correction des mésappariements. Les auteurs rapportent ici que la protéine AtMSH2 partage des propriétés fonctionnelles avec les protéines homologues caractérisées précédemment. Premièrement, la surexpression de cette protéine chez Escherichia coli produit un phénotype mutateur semblable à celui rapporté pour d'autres homologues fonctionnels. Deuxièmement, des expériences de retard sur gel ont révélé que la protéine AtMSH2 a une affinité 10 fois supérieure pour de l'ADN contenant une paire de bases mésappariées par rapport à de l'ADN dont les bases sont parfaitement complémentaires. Ces résultats constituent des évidences expérimentales de l'homologie fonctionnelle entre MutS et son homologue présent chez Arabidopsis thaliana.Mots clés : correction des mésappariements de l'ADN, ADN hétéroduplex, taux de mutation. [ABSTRACT FROM AUTHOR]

We have used transformation of yeast with lincarized plasmids to study the transfer of information to the unbroken chromosome during double-strand break repair. Using a strain which carried the wild-type HIS3 allele, and a linearized plasmid which carried a mutant his3 allele, we have obtained His transformants. In these, double-strand break repair has resulted in precise transfer of genetic information from the plasmid to the chromosome. Such repair events, we suggest, are gene conversions which entail the formation of heteroduplex DNA on the (unbroken) chromosome. If this suggestion is correct, our results reflect the spatial distribution of such heteroduplex DNA. Transfer of information from the plasmid to the chromosome was obtained at a maximal frequency of 1.5% of the repair events, and showed a dependence with distance. Transformation to His was also obtained with a 2-kbp insertion and with a deletion of 200 bp. The latter results suggest that gene conversion of large heterologies can occur via repair of a heteroduplex DNA intermediate. [ABSTRACT FROM AUTHOR]

The frequency of meiotic gene conversion often varies linearly from one end of the gene to the other. This phenomenon has been called 'polarity'. In this review, we will primarily discuss studies of polarity that have been done in the yeast Saccharomyces cerevisiae (ARG4 and HIS4 loci) and in Ascobolus (b2 locus) with an emphasis on possible mechanisms. The genetic and physical data obtained at these 'hotspots' of recombination strongly suggests that the formation of a polarity gradient reflects both the frequency of heteroduplex formation and the processing of this recombination intermediate by mismatch-repair-dependent processes. [ABSTRACT FROM AUTHOR]

For the formation of recombinants involving closely linked markers, two distinct processes play a role. The recombinational interaction between homologous DNA molecules results in the presence of heteroduplex DNA joining the parental components of the recombinant. The presence of markers distinguishing the parents in the region of heteroduplex DNA can result in base pair mismatches. The post recombination repair of such mismatches can contribute to the separation of closely linked markers. The processes responsible for such repair also play roles in mutation avoidance. The specificities, functions and contribution to the formation of recombinants for closely linked markers of the processes in Escherichia coli are described. [ABSTRACT FROM AUTHOR]

Ten codominant RAPD markers, ranging in size from about 300 to about 1350 bp, were identified in mapping populations of chickpea ( Cicer arietinum L.) and diploid strawberry ( Fragaria vesca L.). A distinguishing feature of all ten markers, and perhaps of codominant RAPD markers in general, was the presence in heterozygous individuals of a non-parental, heteroduplex band migrating more slowly than either of the respective parental bands. This non-parental band could also be generated by mixing parental DNAs before PCR (template mixing). As a means of identifying primers likely to detect codominant RAPD markers, parental and mixed-template (parent-parent) PCR-product gel lanes were compared for 20 previously untested RAPD primers (10-base oligomers). Four primers that produced a total of five non-parental, heteroduplex bands in mixed-template reactions were selected, and then used to detect a total of five segregating, codominant markers and nine dominant markers in the respective F mapping population, a codominant marker frequency of 35.7%. When closely migrating fast and slow bands of codominant RAPDs were difficult to differentiate, parent-progeny template mixing was used to deliberately generate heteroduplex bands in fast- or slow-band F homozygotes, respectively, allowing confirmation of marker phenotype. [ABSTRACT FROM AUTHOR]

Mismatch-specific enzymatic activity was sought for in nuclei from normal and transformed plant cells originating from tobacco ( Nicotiana tabacum) callus and crown gall tumor induced by Agrobacterium tumefaciens. The specific enzymatic activity was assayed with substrates derived from synthetic oligonucleotides (19-mer sequences corresponding to the human K-ras gene). Single-base changes in the middle of the sequence were the basis for creating heteroduplexes with all eight mismatches. Homo- and heteroduplexes were ligated in a size ladder and used as substrates. We detected mismatch-specific DNA breakdown and determined basic requirements for the reations. Kinetic analysis indicates the following reactivity order of preference: C : A=C : C=C : T>G : T∼A : A∼G : A∼G : G∼T : T>>G : C. It can be said now that specific mismatch recognition and repair activities have been detected in all kingdoms of living species. [ABSTRACT FROM AUTHOR]

Spontaneous and double-strand break (DSB)-induced gene conversion in Saccharomyces cerevisiae was assayed using non-tandem chromosomal direct repeat crosses and plasmid × chromosome crosses. Each cross involved identical ura3 alleles marked with phenotypically silent restriction fragment length polymorphic (RFLP) mutations at approximately 100-bp intervals. DSBs introduced in vivo at HO sites in one allele stimulated recombination to Ura+ by more than two orders of magnitude. Spontaneous gene-conversion products were isolated from a related strain lacking a functional HO nuclease gene. The multiple markers did not appear to influence the frequency of direct repeat deletions for spontaneous or DSB-induced events. DSB-induced conversion reflected efficient mismatch repair of heteroduplex DNA. Conversion frequencies of equidistant markers on opposites sides of the DSB were similar in the direct repeat cross. In contrast, markers 5′ of the DSB (promoter-proximal) converted more often than 3′ markers in plasmid × chromosome crosses, a possible consequence of crossing-over associated with long conversion tracts. With direct repeats, bidirectional tracts (extending 5′ and 3′ of the DSB) occurred twice as often as in a plasmid × chromosome cross in which DSBs were introduced into the plasmid-borne allele. A key difference between the direct-repeat and plasmid×chromosome crosses is that the ends of a broken plasmid are linked, whereas the ends of a broken chromosome are unlinked. We tested whether linkage of ends influenced tract directionality using a second plasmid × chromosome cross in which DSBs were introduced into the chromosomal allele and found few bidirectional tracts. Thus, chromosome environment, but not linkage of ends, influences tract directionality. The similar tract spectra of the two plasmid × chromosome crosses suggest that similar mechanisms are involved whether recombination is initiated by DSBs in plasmid or chromosomal alleles. [ABSTRACT FROM AUTHOR]

Double-strand break (DSB)-induced gene conversion in yeast was studied in crosses between ura3 heteroalleles carrying phenotypically silent markers at approximately 100-bp intervals, which allow high-resolution analyses of tract structures. DSBs were introduced in vivo by HO nuclease at sites within shared homology and were repaired using information donated by unbroken alleles. Previous studies with these types of crosses showed that most tracts of Ura products are continuous, unidirectional, and extend away from frameshift mutations in donor alleles. Here we demonstrate that biased tract directionality is a consequence of selection pressure against Ura products that results when frameshift mutations in donor alleles are transferred to recipient alleles. We also performed crosses in which frameshift mutations in recipient and donor alleles were arranged such that events initiated at DSBs could not convert broken alleles to Ura via a single gap repair event or a single long-tract mismatch repair event in heteroduplex DNA. This constraint led to low recombination frequencies relative to unconstrained crosses, and inhibited preferential conversion of broken alleles. Physical analysis of 51 DSB-induced products arising from multiple recombinational repair events suggested that hDNA formation is generally limiting, but that some hDNA regions may extend more than 600 bp. Among these products, markers separated by 20 by were independently repaired about 40% of the time. [ABSTRACT FROM AUTHOR]

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2 –/– mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model—asymmetry in recombination intermediates and D-loop migration—may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution. • Fine-scale mapping of heteroduplex DNA in mismatch repair-defective mice • Mismatch repair-mediated heteroduplex rejection is not evident during meiosis • Heteroduplex DNA patterns revise the canonical meiotic recombination model in mice • D-loop migration and asymmetric Holliday junction cleavage are intrinsic features Crossover formation is essential for gamete viability, yet our understanding of mammalian mechanisms largely relies on inference from yeast. Peterson et al. illuminate mechanisms of crossover formation, including commonalities with yeast while distinguishing mammalian-specific differences. This fine-scale study of recombination intermediates allowed revision of the canonical model of crossover formation in mice. [ABSTRACT FROM AUTHOR]

Homologous recombination is an important source of biological genetic variation. Limited by detection methods, there are only a few reports on the homologous recombination in high plants and its product - heteroduplex DNA (hDNA). In the present study, applying the strategy of detecting hDNA by constructing populations from inhibited post-meiotic segregation, two hybrid triploid populations were constructed from two maternal parents inPopulus tomentosa by inhibiting post-meiotic segregation. One hundred and ten simple sequence repeat (SSR) markers were used to study the occurrence and variation of hDNA on nine chromosomes inP. tomentosa with different genotypes. The results showed that the frequencies of hDNA between two female parents inP. tomentosa ranged from 8.5% to 87.2%. The hDNA frequency was positively correlated to the distance from the centromere, but the average hDNA frequency on a chromosome had no correlation with the chromosome length. One to 3 times recombination events were detected on most chromosomes, and only a few four- or five-times recombination events were detected. The overall frequencies of hDNA on the same chromosome in two genotypic individuals were roughly similar, while the hDNA frequencies varied greatly at specific SSR loci. Compared withTacamahaca poplar hybrid,P. pseudo-simonii × P. nigra 'Zheyin3#', detection of homologous recombination times and the frequency and location of hDNA were largely different. This study is the first to describe the characteristics and variations of homologous recombination inP. tomentosa with two different genotypes, which will provide valuable insights for exploring the characteristics and variations of homologous recombination among interspecies and intraspecies in higher plant.

Summary Meiotic recombination is essential for fertility and allelic shuffling. Canonical recombination models fail to capture the observed complexity of meiotic recombinants. Here, by combining genome-wide meiotic heteroduplex DNA patterns with meiotic DNA double-strand break (DSB) sites, we show that part of this complexity results from frequent template switching during synthesis-dependent strand annealing that yields noncrossovers and from branch migration of double Holliday junction (dHJ)-containing intermediates that mainly yield crossovers. This complexity also results from asymmetric positioning of crossover intermediates relative to the initiating DSB and Msh2-independent conversions promoted by the suspected dHJ resolvase Mlh1-3 as well as Exo1 and Sgs1. Finally, we show that dHJ resolution is biased toward cleavage of the pair of strands containing newly synthesized DNA near the junctions and that this bias can be decoupled from the crossover-biased dHJ resolution. These properties are likely conserved in eukaryotes containing ZMM proteins, which includes mammals. [ABSTRACT FROM AUTHOR]

Summary Heteroduplex DNA (hetDNA) is a key molecular intermediate during the repair of mitotic double-strand breaks by homologous recombination, but its relationship to 5′ end resection and/or 3′ end extension is poorly understood. In the current study, we examined how perturbations in these processes affect the hetDNA profile associated with repair of a defined double-strand break (DSB) by the synthesis-dependent strand-annealing (SDSA) pathway. Loss of either the Exo1 or Sgs1 long-range resection pathway significantly shortened hetDNA, suggesting that these pathways normally collaborate during DSB repair. In addition, altering the processivity or proofreading activity of DNA polymerase δ shortened hetDNA length or reduced break-adjacent mismatch removal, respectively, demonstrating that this is the primary polymerase that extends both 3′ ends. Data are most consistent with the extent of DNA synthesis from the invading end being the primary determinant of hetDNA length during SDSA. [ABSTRACT FROM AUTHOR]