Methyl-directed DNA mismatch repair in Escherichia coli

Some of the molecular aspects of methyl-directed mismatch repair in E. coli have been characterized. These include: mismatch recognition by mutS protein in which different mispairs are bound with different affinities; the direct involvement of d(GATC) sites; and strand scission by mutH protein at d(GATC) sequences with strand selection based on methylation of the DNA at those sites. In addition, communication over a distance between a mismatch and d(GATC) sites has been implicated. Analysis of mismatch correction in a defined system (Lahue et al., unpublished) should provide a direct means to further molecular aspects of this process.     

Tyr212: a key residue involved in strand discrimination by the DNA mismatch repair endonuclease MutH

The molecular mechanism of how the dam-methylation status of the DNA is recognized during DNA mismatch repair by the strand discrimination endonuclease MutH is not known. A comparison of the crystal structure of MutH with those of co-crystal structures of several restriction endonucleases, together with a multiple sequence alignment of MutH and related proteins suggested that Phe94, Arg184 and Tyr212 could be involved in discrimination between a methylated or unmethylated adenine in the d(GATC) sequence. A mutational analysis revealed that the variants R184A and Y212S, but not F94A, were substantially reduced in their ability to complement a mismatch repair deficiency in a mutH(-) Escherichia coli strain. In vitro, R184A displayed a strongly reduced endonuclease activity, whereas the Y212S variant has almost completely lost its preference for cleaving the unmethylated strand at hemimethylated d(GATC) sites. Furthermore, the Y212 variant can cleave fully methlyated d(GATC) sites at a comparable rate to unmethylated d(GATC) sites. This demonstrates that Tyr212 is an important, if not the only amino acid residue in MutH for sensing the methylation status of the DNA.     

GATC sequence and mismatch repair in Escherichia coli.

The Escherichia coli mismatch repair system greatly improves DNA replication fidelity by repairing single mispaired and unpaired bases in newly synthesized DNA strands. Transient undermethylation of the GATC sequences makes the newly synthesized strands susceptible to mismatch repair enzymes. The role of unmethylated GATC sequences in mismatch repair was tested in transfection experiments with heteroduplex DNA of phage phi 174 without any GATC sequence or with two GATC sequences, containing in addition either a G:T mismatch (Eam+/Eam3) or a G:A mismatch (Bam+/Bam16). It appears that only DNA containing GATC sequences is subject to efficient mismatch repair dependent on E. coli mutH, mutL, mutS and mutU genes; however, also in the absence of GATC sequence some mut-dependent mismatch repair can be observed. These observations suggest that the mismatch repair enzymes recognize both the mismatch and the unmethylated GATC sequence in DNA over long distances. The presence of GATC sequence(s) in the substrate appears to be required for full mismatch repair activity and not only for its strand specificity according to the GATC methylation state.     

d(GATC) sequences influence Escherichia coli mismatch repair in a distance-dependent manner from positions both upstream and downstream of the mismatch

The role of d(GATC) sites in determining the efficiency of methyl-directed mismatch repair in Escherichia coli was investigated. Transfection of host bacteria, both proficient and deficient in mismatch repair, with a series of artificially constructed M13 heteroduplexes showed that a decrease in the total number of d(GATC) sequences within these vectors lowered the efficiency of repair in vivo. Single hemimethylated d(GATC) sequences were still able to direct the correction event to the unmethylated strand, providing that the mismatch to d(GATC) site distance was shorter than approximately 1 kb. In excess of this distance, the effect of hemimethylated d(GATC) sites on mismatch correction was almost unnoticeable. The directionality of the repair event could be dictated by d(GATC) sequences situated both upstream and downstream of the mispair, suggesting that this important antimutagenic pathway can proceed bidirectionally.     

Initiation of methyl-directed mismatch repair.

Escherichia coli MutH possesses an extremely weak d(GATC) endonuclease that responds to the state of methylation of the sequence (Welsh, K. M., Lu, A.-L., Clark, S., and Modrich, P. (1987) J. Biol. Chem. 262, 15624-15629). MutH endonuclease is activated in a reaction that requires MutS, MutL, ATP, and Mg2+ and depends upon the presence of a mismatch within the DNA. The degree of activation correlates with the efficiency with which a particular mismatch is subject to methyl-directed repair (G-T greater than G-G greater than A-C greater than C-C), and activated MutH responds to the state of DNA adenine methylation. Incision of an unmethylated strand occurs immediately 5' to a d(GATC) sequence, leaving 5' phosphate and 3' hydroxy termini (pN decreases pGpAp-TpC). Unmethylated d(GATC) sites are subject to double strand cleavage by activated MutH, an effect that may account for the killing of dam- mutants by 2-aminopurine. The mechanism of activation apparently requires ATP hydrolysis since adenosine-5'-O-(3-thiotriphosphate) not only fails to support the reaction but also inhibits activation promoted by ATP. The process has no obligate polarity as d(GATC) site incision by the activated nuclease can occur either 3' or 5' to the mismatch on an unmethylated strand. However, activation is sensitive to DNA topology. Circular heteroduplexes are better substrates than linear molecules, and activity of DNAs of the latter class depends on placement of the mismatch and d(GATC) site within the molecule. MutH activation is supported by a 6-kilobase linear heteroduplex in which the mismatch and d(GATC) site are centrally located and separated by 1 kilobase, but a related molecule, in which the two sites are located near opposite ends of the DNA, is essentially inactive as substrate. We conclude that MutH activation represents the initiation stage of methyl-directed repair and suggest that interaction of a mismatch and a d(GATC) site is provoked by MutS binding to a mispair, with subsequent ATP-dependent translocation of one or more Mut proteins along the helix leading to cleavage at a d(GATC) sequence on either side of the mismatch.     

Processing of mispaired and unpaired bases in heteroduplex DNA in E. coli

Bacteriophage lambda and phi X 174 DNAs, carrying sequenced mutations, have been used to construct in vitro defined species of heteroduplex DNA. Such heteroduplex DNAs were introduced by transfection, as single copies, into E. coli host cells. The progeny of individual heteroduplex molecules from each infective center was analyzed. The effect of the presence of GATC sequences (phi X 174 system) and of their methylation (lambda system) was tested. The following conclusions can be drawn: some mismatched base pairs trigger the process of mismatch repair, causing a localized strand-to-strand information transfer in heteroduplex DNA: transition mismatches G:T and A:C are efficiently repaired, whereas the six transversion mismatches are not always readily recognized and/or repaired. The recognition of transversion mismatches appears to depend on the neighbouring nucleotide sequence; single unpaired bases (frameshift mutation "mismatches") are recognized and repaired, some equally efficiently on both strands (longer and shorter), some more efficiently on the shorter (-1) strand; large non-homologies (about 800 bases) are not repaired by the Mut H, L, S, U system, but some other process repairs the non-homology with a relatively low efficiency; full methylation of GATC sequences inhibits mismatch repair on the methylated strand: this is the chemical basis of strand discrimination (old/new) in mismatch correction; unmethylated GATC sequences appear to improve mismatch repair of a G:T mismatch in phi X 174 DNA, but there may be some residual mismatch repair in GATC-free phi X 174, at least for some mismatches.(ABSTRACT TRUNCATED AT 250 WORDS)     

GATC sequences, DNA nicks and the MutH function in Escherichia coli mismatch repair.

Circular heteroduplex DNAs of bacteriophage phi X174 have been constructed carrying either a G:T (Eam+/Eam3) or a G:A (Bam+/Bam16) mismatch and containing either two, one or no GATC sequences. Mismatches were efficiently repaired in wild-type Escherichia coli transfected with phi X174 heteroduplexes only when two unmethylated GATC sequences were present in phi X174 DNA. The requirements for GATC sequences in substrate DNA and for the E. coli MutH function in E. coli mismatch repair can be alleviated by the presence of a persistent nick (transfection with nicked heteroduplex DNA in ligase temperature-sensitive mutant at 40 degrees C). A persistent nick in the GATC sequence is as effective in stimulating mutL- and mutS-dependent mismatch repair as a nick distant from the GATC sequence and from the mismatch. These observations suggest that the MutH protein participates in methyl-directed mismatch repair by recognizing unmethylated DNA GATC sequences and/or stimulating the nicking of unmethylated strands.     

Sequence and substrate specificity of isolated DNA methylases from Escherichia coli C

Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.     

Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis

A covalently closed, circular heteroduplex containing a G-T mismatch and a single hemimethylated d(GATC) site is subject to efficient methyl-directed mismatch correction in Escherichia coli extracts when repair DNA synthesis is severely restricted by limiting the concentration of exogenously supplied deoxyribonucleoside-5'-triphosphates or by supplementing reactions with chain-terminating 2',3'-dideoxynucleoside triphosphates. However, repair under these conditions results in formation of a single-strand gap in the region of the molecule containing the mismatch and the d(GATC) site. These findings indicate that repair DNA synthesis required for methyl-directed correction can initiate in the vicinity of the mispair, and they are most consistent with a repair reaction involving 3'----5' excision (or strand displacement) from the d(GATC) site followed by 5'----3' repair DNA synthesis initiating in the vicinity of the mismatch.     

Structural basis for MutH activation in E.coli mismatch repair and relationship of MutH to restriction endonucleases.

MutS, MutL and MutH are the three essential proteins for initiation of methyl-directed DNA mismatch repair to correct mistakes made during DNA replication in Escherichia coli. MutH cleaves a newly synthesized and unmethylated daughter strand 5' to the sequence d(GATC) in a hemi-methylated duplex. Activation of MutH requires the recognition of a DNA mismatch by MutS and MutL. We have crystallized MutH in two space groups and solved the structures at 1.7 and 2.3 A resolution, respectively. The active site of MutH is located at an interface between two subdomains that pivot relative to one another, as revealed by comparison of the crystal structures, and this presumably regulates the nuclease activity. The relative motion of the two subdomains in MutH correlates with the position of a protruding C-terminal helix. This helix appears to act as a molecular lever through which MutS and MutL may communicate the detection of a DNA mismatch and activate MutH. With sequence homology to Sau3AI and structural similarity to PvuII endonuclease, MutH is clearly related to these enzymes by divergent evolution, and this suggests that type II restriction endonucleases evolved from a common ancestor.     

GATC flanking sequences regulate Dam activity: evidence for how Dam specificity may influence pap expression

Escherichia coli DNA adenine methyltransferase (Dam) plays essential roles in DNA replication, mismatch repair and gene regulation. The differential methylation by Dam of the two GATC sequences in the pap promoter regulates the expression of pili genes necessary for uropathogenic E.coli cellular adhesion. Dam processively methylates GATC sites in various DNA substrates, yet the two pap GATC sites are not processively methylated. We previously proposed that the flanking sequences surrounding the two pap GATC sites contribute to the enzyme's distributive methylation. We show here that replacement of the poorly methylated pap GATC sites with sites predicted to be processively methylated indeed results in an increase in Dam processivity. The increased processivity is due to a change in the methyltransfer kinetics and not the binding efficiency of Dam. A competition experiment in which the flanking sequences of only one pap GATC site were altered demonstrates that the GATC flanking sequences directly regulate the enzyme's catalytic efficiency. The GATC flanking sequences in Dam-regulated promoters in E.coli and other bacteria are similar to those in the pap promoter. Gene regulation from some of these promoters involves mechanisms and proteins that are quite different from those in the pap operon. Further, GATC sequences previously identified to remain unmethylated within the E.coli genome, but whose function remains largely unassigned, are flanked by sequences predicted to be poorly methylated. We conclude that the GATC flanking sequences may be critical for expression of pap and other Dam-regulated genes by affecting the activity of Dam at such sites and, thus, its processivity. A model is proposed, illustrating how the sequences flanking the GATC sites in Dam-regulated promoters may contribute to this epigenetic mechanism of gene expression, and how flanking sequences contribute to the diverse biological roles of Dam.     

Mechanism and control of interspecies recombination in Escherichia coli. I. Mismatch repair,methylation, recombination and replication functions.

A genetic analysis of interspecies recombination in Escherichia coli between the linear Hfr DNA from Salmonella typhimurium and the circular recipient chromosome reveals some fundamental aspects of recombination between related DNA sequences. The MutS and MutL mismatch binding proteins edit (prevent) homeologous recombination between these 16% diverged genomes by at least two distinct mechanisms. One is MutH independent and presumably acts by aborting the initiated recombination through the UvrD helicase activity. The RecBCD nuclease might contribute to this editing step, presumably by preventing reiterated initiations of recombination at a given locus. The other editing mechanism is MutH dependent, requires unmethylated GATC sequences, and probably corresponds to an incomplete long-patch mismatch repair process that does not depend on UvrD helicase activity. Insignificant effects of the Dam methylation of parental DNAs suggest that unmethylated GATC sequences involved in the MutH-dependent editing are newly synthesized in the course of recombination. This hypothetical, recombination-associated DNA synthesis involves PriA and RecF functions, which, therefore, determine the extent of MutH effect on interspecies recombination. Sequence divergence of recombining DNAs appears to limit the frequency, length, and stability of early heteroduplex intermediates, which can be stabilized, and the recombinants mature via the initiation of DNA replication.     

Effect of uracil situated in the vicinity of a mispair on the directionality of mismatch correction in Escherichia coli.

We wanted to establish whether strand breaks and gaps, arising during the removal of uracil from newly-synthesized DNA, can be utilized as strand discrimination signals by the methyl-directed mismatch repair system of Escherichia coli. For this purpose, we constructed a series of M13 heteroduplexes that contained a single uracil residue situated either upstream or downstream from a G/T or an A/C mispair. Transfections of these constructs into E. coli strains, either proficient of deficient in mismatch or uracil repair, allowed us to follow the fate of these mispairs in vivo. Our data show that the intermediates of uracil repair cannot substitute for the strand-discrimination signals generated by the MutH protein, which is thought to initiate the methyl-directed mismatch repair process by nicking the unmethylated strand of a newly-synthesized DNA duplex at d(GATC) sites. However, processing of uracil residues situated upstream from the mispair was shown to reduce the yield of the progeny phage arising from the uracil-containing strand, presumably as a result of co-repair of the base analogue and the mispair.     

The function of Asp70, Glu77 and Lys79 in the Escherichia coli MutH protein

The MutH protein, which is part of the Dam-directed mismatch repair system of Escherichia coli, introduces nicks in the unmethylated strand of a hemi-methylated DNA duplex. The latent endonuclease activity of MutH is activated by interaction with MutL, another member of the repair system. The crystal structure of MutH suggested that the active site residues include Asp70, Glu77 and Lys79, which are located at the bottom of a cleft where DNA binding probably occurs. We mutated these residues to alanines and found that the mutant proteins were unable to complement a chromosomal mutH deletion. The purified mutant proteins were able to bind to DNA with a hemi-methylated GATC sequence but had no detectable endonuclease activity with or without MutL. Although the data are consistent with the prediction of a catalytic role for Asp70, Glu77 and Lys79, it cannot be excluded that they are also involved in binding to MutL.     

DNA loop repair by Escherichia coli cell extracts

The nick-directed DNA repair efficiency of a set of M13mp18-derived heteroduplexes containing 8-, 12-, 16-, 22-, 27-, 45-, and 429-nucleotide loops was determined by in vitro assay. Unpaired nucleotides of each heteroduplex reside within overlapping recognition sites for two restriction endonucleases, permitting independent evaluation of repair occurring on either DNA strand. Our results show that a strand break located either 3' or 5' to the loop is sufficient to direct heterology repair to the nicked strand in Escherichia coli extracts. Strand-specific repair by this system requires Mg2+ and the four dNTPs and is equally efficient on insertions and deletions. This activity is distinct from the MutHLS mismatch repair pathway. Strand specificity and repair efficiency are largely independent of the GATC methylation state of the DNA and presence of the products of mismatch repair genes mutH, mutL, and mutS. This study provides evidence for a loop repair pathway in E. coli that is distinct from conventional mismatch repair.     

Repair of Single Base-Pair Transversion Mismatches of Escherichia Coli in Vitro: Correction of Certain a/G Mismatches Is Independent of Dam Methylation and Host Muthls Gene Functions

Six different base-pair transversion mismatches are repaired with different efficiencies in an in vitro mismatch repair system. In particular, the T/T and C/C mismatches appear to be less efficiently repaired than the A/A and G/G mismatches. Four A/G and four C/T mismatches at different positions are repaired to different extents. One of the A/G mismatches is repaired equally efficiently when DNA heteroduplexes are fully methylated or hemi-methylated at the d(GATC) sequences. This type of mismatch repair appears to be unidirectional with A to C conversion by acting at A/G mispairs to restore the C/G pairs. This methylation-independent correction is not controlled by the mutH, mutL, mutS, uvrE, uvrB, phr, recA, recF, and recJ gene products. The independence of the transversion mismatch repair of these genes and methylation distinguishes this from the known mismatch repair pathways.     

Methylation of GATC sites is required for precise timing between rounds of DNA replication in Escherichia coli.

We have used the Koppes and Nordstr?m (Cell 44:117-124, 1986) CsCl density transfer approach for analysis of DNA from exponentially growing, isogenic Escherichia coli dam+ and dam mutant cells to show that timing between DNA replication initiation events is precise in the dam+ cells but is essentially random in the dam cells. Thus, methylation of one or more GATC sites, such as those found in unusual abundance within the origin, oriC, is required for precise timing between rounds of DNA replication, and precise timing between initiation events is not required for cell viability. Both the dam-3 point mutant and the delta(dam)100 complete deletion mutant were examined. The results were independent of the mismatch repair system; E. coli mutH cells showed precise timing, whereas timing in the isogenic E. coli mutH delta(dam)100 double mutant was random. The mechanism is thus different from the role of Dam methylation in mismatch repair and probably involves conversion of hemimethylated GATC sites present in daughter origins just after initiation to a fully methylated state.     

Endonucleolytic function of MutLalpha in human mismatch repair

Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLalpha (MLH1*PMS2 heterodimer). MutLalpha is required for mismatch repair, but its function in this process is unclear. We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5'-to-3' activity of MutSalpha-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.