Influence of DNA sequence identity on efficiency of targeted gene replacement.

We have developed a system for analyzing recombination between a DNA fragment released in the nucleus from a single-copy plasmid and a genomic target in order to determine the influence of DNA sequence mismatches on the frequency of gene replacement in Saccharomyces cerevisiae. Mismatching was shown to be a potent barrier to efficient gene replacement, but its effect was considerably ameliorated by the presence of DNA sequences that are identical to the genomic target at one end of a chimeric DNA fragment. Disruption of the mismatch repair gene MSH2 greatly reduces but does not eliminate the barrier to recombination between mismatched DNA fragment and genomic target sequences, indicating that the inhibition of gene replacement with mismatched sequences is at least partially under the control of mismatch repair. We also found that mismatched sequences inhibited recombination between a DNA fragment and the genome only when they were close to the edge of the fragment. Together these data indicate that while mismatches can destabilize the relationship between a DNA fragment and a genomic target sequence, they will only do so if they are likely to be in the heteroduplex formed between the recombining molecules.     

Multicopy single-stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. In Escherichia coli , such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli . To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.     

Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA.

The independent repair of mismatched nucleotides present in heteroduplex DNA has been used to explain gene conversion and map expansion after general genetic recombination. We have constructed and purified heteroduplex plasmid DNAs that contain heteroallelic 10-base-pair insertion-deletion mismatches. These DNA substrates are similar in structure to the heteroduplex DNA intermediates that have been proposed to be produced during the genetic recombination of plasmids. These DNA substrates were transformed into wild-type and mutant Escherichia coli strains, and the fate of the heteroduplex DNA was determined by both restriction mapping and genetic tests. Independent repair events that yielded a wild-type Tetr gene were observed at a frequency of approximately 1% in both wild-type and recB recC sbcB mutant E. coli strains. The independent repair of small insertion-deletion-type mismatches separated by 1,243 base pairs was found to be reduced by recF, recJ, and ssb single mutations in an otherwise wild-type genetic background and reduced by recF, recJ, and recO mutations in a recB recC sbcB genetic background (the ssb mutation was not tested in the latter background). Independent repair of small insertion-deletion-type mismatched nucleotides that were as close as 312 nucleotides apart was observed. There was no apparent bias in favor of the insertion or deletion of mutant sequences.     

Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair.

Sequence homology is expected to influence recombination. To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog. Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions. Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold. Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing. For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair. Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination. Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination. The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis.     

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral introduction of UV damage can potentially compromise genetic interpretations.     

Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.     

Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: cloning and expression of the hexA gene.

Mutations affecting heteroduplex DNA mismatch repair in Streptococcus pneumoniae were localized in two genes, hexA and hexB, by fractionation of restriction fragments carrying mutant alleles. A fragment containing the hexA4 allele was cloned in the S. pneumoniae cloning system, and the hexA+ allele was introduced into the recombinant plasmid by chromosomal facilitation of plasmid transfer. Subcloning localized the functional hexA gene to a 3.5-kilobase segment of the cloned pneumococcal DNA. The product of this gene was shown in Bacillus subtilis minicells to be a polypeptide with an Mr of 86,000. Two mutant alleles of hexA showed partial expression of the repair system when present in multicopy plasmids. A model for mismatch repair, which depends on the interaction of two protein components to recognize the mismatched base pair and excise a segment of DNA between strand breaks surrounding the mismatch, is proposed.     

DNA mismatch repair detected in human cell extracts.

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.     

Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

The role of AtMSH2 in homologous recombination in Arabidopsis thaliana

During homologous recombination (HR), a heteroduplex DNA is formed as a consequence of strand invasion. When the two homologous strands differ in sequence, a mismatch is generated. Earlier studies showed that mismatched heteroduplex often triggers abortion of recombination and that a pivotal component of this pathway is the mismatch repair Msh2 protein. In this study, we analysed the roles of AtMSH2 in suppression of recombination in Arabidopsis. We report that AtMSH2 has a broad range of anti-recombination effects: it suppresses recombination between divergent direct repeats in somatic cells or between homologues from different ecotypes during meiosis. This is the first example of a plant gene that affects HR as a function of sequence divergence and that has an anti-recombination meiotic effect. We discuss the implications of these results for plant improvement by gene transfer across species.     

The recombination, repair and modification of DNA

This article is an overview of the author's involvement in theoretical and experimental research on genetic recombination and DNA repair, and also on the enzymic modification of cytosine in DNA to 5-methyl cytosine. It includes the history of the discovery of the central intermediate in genetic recombination at the DNA level, and the repair of mismatched bases. These explain the major features of genetic fine structure. The first repair and recombination defective mutants in any eukaryote were isolated in the smut fungus Ustilago maydis. The hypothesis that DNA methylation has a role in gene expression in higher organism is now supported by abundant evidence. Direct evidence that gene silencing in mammalian cells is causally related to DNA methylation has been obtained.     

Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells

Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93-101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2(-/-) cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.     

Gene conversion in Escherichia coli. Resolution of heteroallelic mismatched nucleotides by co-repair

We have constructed heteroduplex plasmid DNA that is similar in structure to the heteroduplex DNA expected to be produced during genetic recombination of plasmids, and studied its repair after transformation into different Escherichia coli strains. The heteroduplex DNA was constructed using two different parental plasmids, each of which contained a different ten-nucleotide insertion mutation. The effect of different defined states of dam-methylation on repair was also examined. We found that heteroduplex DNA repair occurred prior to the replication of the substrate DNA 60 to 80% of the time, regardless of the state of DNA methylation. Most excision/synthesis tracts covered two markers separated by 1243 base-pairs, and this process has been termed co-repair. The most efficient co-repair pathway was the Dam-instructed repair pathway that required the mutH, mutL, mutS and uvrD gene products and preferentially used the methylated strand as the template for DNA synthesis. If there was no methylation asymmetry, mismatch nucleotide repair occurred with a similar frequency; however, no strand bias was observed. Co-repair of symmetrically methylated heteroduplex DNA required the mutS and uvrD gene products, while repair of unmethylated heteroduplex DNA also required the mutL and mutH gene products.     

Single-molecule views of MutS on mismatched DNA

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.     

Targeted gene repair and its application to neurodegenerative disorders

Synthetic DNA oligonucleotides can direct the exchange of single nucleotides within coding regions of mammalian genes by hybridizing to their complementary sequence in the chromosome and creating a recombination joint structure with a single mismatched base pair. Inherent DNA repair processes recognize the mismatch and resolve it using the DNA sequence of the oligonucleotide vector as the template. This gene surgery approach can be used to repair mutations or to disrupt tri-nucleotide repeats in dysfunctional genes responsible for neurological disorders.     

DNA binding and protein-protein interaction sites in MutS, a mismatched DNA recognition protein from Thermus thermophilus HB8

The mismatch repair system repairs mismatched base pairs, which are caused by either DNA replication errors, DNA damage, or genetic recombination. Mismatch repair begins with the recognition of mismatched base pairs in DNA by MutS. Protein denaturation and limited proteolysis experiments suggest that Thermus thermophilus MutS can be divided into three structural domains as follows: A (N-terminal domain), B (central domain), and C (C-terminal domain) (Tachiki, H., Kato, R., Masui, R., Hasegawa, K., Itakura, H., Fukuyama, K., and Kuramitsu, S. (1998) Nucleic Acids Res. 26, 4153-4159). To investigate the functions of each domain in detail, truncated genes corresponding to the domains were designed. The gene products were overproduced in Escherichia coli, purified, and assayed for various activities. The MutS-MutS protein interaction site was determined by size-exclusion chromatography to be located in the B domain. The B domain was also found to possess nonspecific double-stranded DNA-binding ability. The C domain, which contains a Walker's A-type nucleotide-binding motif, demonstrated ATPase activity and specific DNA recognition of mismatched base pairs. These ATPase and specific DNA binding activities were found to be dependent upon C domain dimerization.     

ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.     

Marker-Dependent Recombination in T4 Bacteriophage. III. Structural Prerequisites for Marker Discrimination

Distance- as well as marker-dependence of genetic recombination of bacteriophage T4 was studied in crosses between rIIB mutants with known base sequences. The notion of a "basic recombination," which is the recombination within distances shorter than hybrid DNA length in the absence of mismatch repair and any marker effects, was substantiated. The basic recombination frequency per base pair can serve as an objective parameter (natural constant) of general recombination reflecting its intensity. Comparative studies of the recombination properties of rIIB mutants with various sequence changes in the mutated sites showed that the main factor determining the probability of mismatch repair in recombination heteroduplexes is the length of a continuous heterologous region. A run of A:T pairs immediately adjoining the mismatch appears to stimulate its repair. In the case of mismatches with DNA strands of unequal length, formed by frameshift mutations, the repair is asymmetric, the longer strand (bulge) being preferentially removed. A pathway for mismatch repair including sequential action of endonuclease VII (gp49)----3'----5' exonuclease (gp43)----DNA polymerase (gp43)----DNA ligase (gp30) was proposed. A possible identity of the recombinational mismatch repair mechanism to that operating to produce mutations via sequence conversion is discussed.