Prokaryotic and eukaryotic cells are capable of repairing mismatched base pairs in their DNA. Mismatched base pairs in DNA can arise by several processes. One of the most important is by replication errors. In this case, the correct base of the mismatched base pair is located in the parental strand of the newly replicated DNA, and proper correction of the mismatch contributes to the maintenance of the fidelity of the genetic information. A model of mismatch repair of newly synthesized DNA is shown in Figure K1.
A key event in the elucidation of the mechanism of methyl-directed mismatch repair was the development of an assay that allowed mismatch repair to be detected in crude extracts of E. coli (Figure K2). As summarized in the Table, methyl-directed mismatch repair of this heteroduplex substrate was observed in an extract from wildtype (mut+) E. coli, but not in extracts prepared from mutH, mutL, mutS, and uvrD mutants of E. coli. However, biochemical complementation to restore mismatch repair could be achieved by mixing extracts of two different mutator mutants.
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Further experiments showed that repair required excision and resynthesis of DNA. The polymerase required is DNA polymerase III since extracts from a polA strain (deficient in DNA polymerase I) were proficient for mismatch repair, but extracts from a dnaZ strain (deficient for tau and gamma subunits of DNA polymerase III) were not. Using a substrate heteroduplex with a single GATC site allowed mapping of the MutH cleavage site: cleavage occurs on the unmethylated strand immediately 5' to the GATC sequence, leaving a 3'-hydroxyl terminus and a 5'-phosphate (...pN-3'-OH + pGpApTpC....). The observation that MutH can cleave a hemimethylated GATC site located either 3' or 5' to the mismatch suggested that mismatch repair can be initiated by a single-strand break in the unmethylated strand that is located either 3' or 5' of the lesion. This was tested by examining the excision tracts directly by electron microscopy (Figure K3). Regardless of which strand was methylated, the gap was found to span the shortest path between the GATC site and the mismatch. This means that excision must have occurred in a 3'-to-5' direction in one case and a 5'-to-3' direction in the other.
The interaction of E. coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction. Figure 25.16 is a corrected version of the textbook diagram of loop formation (the original figure has several severe mistakes!!!!); a more recent model is shown in Figure K5.
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| dam methylase (DNA adenine methylase) | Methylates adenine to create 6-methyladenine in the sequence GATC. In wildtype E. coli cells, the DNA is normally fully methylated (both strands are methylated). Newly synthesized strands during DNA replication are transiently (that is, for a short period of time) unmethylated, and DNA with one strand methylated and the other strand unmethylated is called hemi-methylated. dam methylase quickly methylates hemi-methylated DNA. Fully unmethylated DNA only occurs in cells lacking dam methylase activity (that is, dam- mutants). dam- mutants have increased rates of mutation (mutator phenotype) and increased recombination frequencies. |
| MutH | Endonuclease that cleaves unmethylated strand just 5' to the G in the sequence GATC (that is, N | GATC) leaving a 3'-OH and 5'-P at the cleavage site. Requires MutL and MutS to activate latent endonuclease activity. |
| MutL | Adds to complex of MutS at mismatch in ATP dependent (but not hydrolysis dependent) step. Acts as a "molecular matchmaker" and uses ATP hydrolysis to bring MutS and MutH together and to stimulate MutH endonuclease activity. Also binds to and loads helicase II. |
| MutS | Binds to all mismatches except C-C; also binds to small insertion or deletion mismatches in which one strand contains one, two, or three extra nucleotides; heteroduplexes with four extra nucleotides are weakly repaired, but larger heterologies do not appear to be recognized. |
| helicase II | Also known as the mutU/uvrD gene product. Requires MutS and MutL to load on at the endonucleolytic cleavage site ("nick"). Moves along a DNA strand in the 3'-to-5' direction. Unwinds the incised strand to make it sensitive to the appropriate single-strand specific exonuclease activity. |
| exonuclease VII | Also known as the xseA gene product. Hydrolyzes single-stranded DNA in the 5'-to-3' direction. |
| RecJ | Hydrolyzes single-stranded DNA in the 5'-to-3' direction. |
| exonuclease I | Also known as the sbcB or xonA gene product. Hydrolyzes single-stranded DNA in the 3'-to-5' direction. |
| DNA polymerase III holoenzyme | The replicative DNA polymerase in E. coli. |
| SSB | Single strand binding protein. |
| DNA ligase | Uses NAD+ to form phosphodiester bonds at "nicks". |
For use of BB492/592 students only. Exerpted with modifications from P. Modrich and R. Lahue (1996) Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu. Rev. Biochem. 65: 101-133.
Mismatch repair stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent sequences. The reaction responsible for strand-specific correction of mispaired bases has been highly conserved during evolution, and homologs of bacterial mutS and mutL, which play key roles in mismatch recognition and initiation of repair, have been identified in yeast and mammalian cells. Inactivation of genes encoding these activities results in a large increase in spontaneous mutability, and in the case of mice and men, predisposition to tumor development.
Bacteria and eukaryotic cells possess several distinct mismatch repair pathways, but we intend to focus on the MutS- and MutL-dependent, so-called long-patch system. This pathway is characterized by broad mismatch specificity and is believed to be responsible for correcting DNA biosyntheic errors and processing recombination heteroduplexes that contain mismatched base pairs.
The prototypic long-patch mismatch correction system is the E. coli methyl-directed pathway, the inactivation of which results in a strong mutator phenotype. Biological observations indicating action of the system on newly replicated DNA confirmed the suspicion that this pathway stabilizes the bacterial genome by correcting DNA biosynthetic errors. Figure AT illustrates the mechanism of the mismatch-provoked methyl-directed excision reaction.
The strand specificity necessary for repair of DNA biosynthetic errors is provided by patterns of adenine methylation in d(GATC) sequences. Since this is a postsynthetic modification, recently synthesized sequences exist in a transiently unmodified state, and the absence of methylation on newly synthesized DNA targets correction to this strand. The methyl-directed pathway has broad mismatch specificity. Although the efficiency of repair of certain transversion mismatches can depend on sequence context, the only base-base mispair for which correction has not been reported is C-C. The system also repairs small insertion/deletion mismatches in which one strand contains one, two, or three extra nucleotides; heteroduplexes with four extra nucleotides are weakly repaired, but larger heterologies do not appear to be recognized.
A single d(GATC) sequence is sufficient to direct mismatch repair. The distance separating the strand signal and the mismatch can be substantial: A d(GATC) site can direct correction of a mispair a kilobase (kb) away, but the strength of the strand signal is greatly reduced when separation distance exceeds two kb. As shown in Figure AT, methyl-directed repair initiates via a mismatch-provoked incision of the unmethylated strand at a d(GATC) sequence in a reaction that requires the mutS, mutL, and mutH gene products and is dependent on ATP hydrolysis. MutS, which exists in solution as oligomers of a 95 kD polypeptide, binds to the mismatch, and MutL, a homodimer of a 68 kD polypeptide, adds to this complex in a reaction that depends on ATP but not on ATP hydrolysis. Interaction of MutS and MutL with the heteroduplex activates a latent endonuclease associated with the 25 kD MutH protein, which cleaves the unmethylated strand at a d(GATC) site. The resulting strand break apparently serves as the primary signal that directs correction to the unmethylated strand, because model heteroduplexes that contain a strand-specific incision, but lack a d(GATC) sequence, are subject to a nick-directed MutH-independent reaction that otherwise appears identical to methyl-directed repair. This finding suggests that strand discontinuities, other than those generated by d(GATC) cleavage, might serve to target mismatch repair to new DNA strands, and evidence for involvement of supplemental signals in mutation avoidance is available. The nature of the additional strand signal(s) has not been determined, but the 3'-terminus of the leading strand or discontinuities on the lagging strand might serve in this regard.
Excision, which is strictly exonucleolytic, initiates at the strand break and proceeds toward the mismatch to terminate at several discrete sites beyond the mispair (Figure AT). A surprising feature of methyl-directed repair is that the strand signal may reside on either side of the mismatch, reflecting a bidirectional capability of the system. Excision initiating from either side of the mismatch requires MutS, MutL, and the mutU/uvrD gene product helicase II, but distinct exonucleases are required in the two cases. Excision from the 5' side of the mispair depends on RecJ exonuclease or exonuclease VII, both of which possess 5'-to-3' hydrolytic activity, whereas excision from the 3' side depends on exonuclease I, which hydrolyzes DNA with 3'-to-5' directionality. Inasmuch as each of these exonucleases is single-strand specific, helicase II is thought to unwind the incised strand in order to render it sensitive to the appropriate exonuclease. Since helicase II is loaded into the heteroduplex at the site of the strand break in a reaction dependent on MutS and MutL, excision may involve concerted unwinding and hydrolysis.
The last step of the methyl-directed reaction is gap repair by DNA polymerase III holoenzyme and ligation of the repair product. The requirement for polymerase III holoenzyme in the purified system is quite specific because DNA polymerase I, T7 DNA polymerase, T4 DNA polymerase, and AMV reverse transcriptase will not substitute. The molecular basis of this specificity for the replication polymerase is not understood but could be indicative of a special affiliation of the mismatch repair system with the replication apparatus.