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DNA Mismatch Repair and Genetic Stability
Summary: Paul Modrich studies mutation avoidance systems that ensure the genetic integrity of a cell.
Mismatch repair rectifies base-pairing errors within the DNA helix. Although a mismatched base pair is a rare occurrence in DNA, inactivation of this DNA repair system has profound consequences for a living cell: a 100- to 1,000-fold increase in the rate of mutation production. Inactivation of the human mismatch repair system and the ensuing genetic instability is the cause of a common form of hereditary colon cancer (HNPCC) and may also play a role in the development of 15–25 percent of sporadic tumors that can occur in a variety of tissues. In bacteria, mismatch repair defects have been linked to the evolutionary development of human pathogens.
Correction of DNA Replication Errors in Escherichia coli
Mismatch repair stabilizes the cellular genome in several ways. The best understood of these is its function as a molecular editor that corrects errors occurring during the course of chromosome replication. Because replication errors are composed of incorrectly paired but otherwise normal Watson-Crick bases, mismatch repair systems rely on secondary signals to direct repair to the newly synthesized DNA strand that contains the replication error. In E. coli, these strand signals are based on methylation at GATC sequences by the DNA adenine methylase. Newly synthesized DNA is transiently unmethylated at GATC sequences, and it is the absence of this modification that directs repair to the new strand.
We have shown that E. coli methyl-directed repair depends on 11 activities, including the products of the four mutator genes: mutH, mutL, mutS, and mutU. MutS is responsible for mismatch recognition, and MutL couples mismatch recognition by MutS to activation of downstream activities. One downstream activity is MutH, an endonuclease that incises the unmethylated strand at a GATC sequence in newly synthesized DNA. MutS and MutL also activate the excision system, which loads at the MutH strand break and is composed of the mutU gene product DNA helicase II and several single-strand-specific exonucleases. The strand break introduced by MutH can be located either 3' or 5' to the mismatch, and MutS and MutL coordinate recognition of these two DNA sites in a manner that permits orientation-dependent loading of the appropriate 3'-to-5' or 5'-to-3' hydrolytic system. Excision terminates upon mismatch removal, and DNA polymerase III holoenzyme repairs the ensuing gap.
Strand-Specific Mismatch Repair in Eukaryotic Cells
In contrast to E. coli, DNA methylation is not believed to play a role in eukaryotic mismatch repair, and the eukaryotic organisms examined to date do not possess a MutH activity. Nevertheless, a DNA strand break is sufficient to confer strand specificity on mismatch repair in human cell extracts, and as in the E. coli reaction, the strand break that directs the reaction may reside 3' or 5' to the mismatch. The human pathway also depends on several activities that are structural and functional homologs of bacterial MutS and MutL. MutSα (a heterodimer of the MutS homologs MSH2 and MSH6) is responsible for most mismatch recognition events in human cells and recruits MutLα (a heterodimer of the MutL homologs MLH1 and PMS2) to mismatch-containing heteroduplex DNA. MutSα and MutLα are of particular interest because genetic inactivation of any of the four polypeptides that comprise these two activities causes cancer.
We have recently reconstituted 3'- and 5'-directed mismatch repair using a set of purified human proteins that includes MutSα, MutLα, Exo1 (an exonuclease that hydrolyzes duplex DNA with 5'-to-3' polarity), RPA (a protein that binds to and stabilizes single-stranded DNA), PCNA (a trimeric protein that forms a clamp-like structure around the DNA molecule), RFC (the enzyme that loads the PCNA clamp onto the helix), and DNA polymerase δ (an enzyme that plays a major role in chromosome replication). Analysis of reactions supported by subsets of these proteins has clarified the mechanism of mismatch repair by this seven-protein system.
Mismatch repair directed by a 5'-strand break occurs by the set of reactions shown in Figure 1 (left panel). Mismatch recognition by MutSα activates the 5'-to-3' hydrolytic activity of Exo1, which loads at the 5'-strand break. RPA controls 5'-to-3' excision by the MutSα•Exo1 complex, leading to termination of hydrolysis upon mismatch removal. Although not essential for excision in this system, MutLα does enhance the mismatch dependence of Exo1 hydrolysis. Resynthesis of the excised DNA segment and restoration of genetic integrity to the helix is mediated by DNA polymerase δ in a reaction that also requires PCNA and RFC.
Mismatch repair directed by a 3'-strand break is more complex. In this case, excision requires MutSα, MutLα, ExoI, RPA, PCNA, and RFC (PCNA and RFC, in addition to DNA polymerase δ, are required for the repair synthesis step as well; Figure 1, right panel). The ability of this six-component system to support 3'-directed excision was surprising because the only DNA hydrolytic activity present was Exo1, which degrades DNA with 5'-to-3' polarity. This paradox was resolved by the demonstration that MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, and PCNA-dependent fashion. Incision of a nicked heteroduplex by activated MutLα endonuclease is strongly biased to the nicked DNA strand and, for molecules with a nick-mismatch separation distance of less than 150 base pairs, tends to occur on the distal side of the mismatch relative to the location of the original strand break that directs the reaction. In a 3' heteroduplex, this results in the introduction of a new strand break on the 5' side of the mispair (Figure 1, right panel). This 5'-strand break serves as an entry site for MutSα-activated Exo1, which removes the mismatch by a 5'-to-3' hydrolytic reaction analogous to that shown in the left panel of Figure 1.
We have localized the probable active site of human MutLα endonuclease to a DQHA(X)2E(X)4E amino acid sequence motif within the PMS2 subunit of MutLα and have found that genetic alteration of this motif abolishes MutLα endonuclease activity in vitro. The DQHA(X)2E(X)4E motif is highly conserved in eukaryotic PMS2 homologs and is also found in archaeal and eubacterial MutL proteins, but it is conspicuously absent in MutL proteins from bacteria such as E. coli that rely on GATC methylation to direct mismatch repair. Collaborative studies with the Thomas Kunkel (National Institute of Environmental Health Sciences) and Michael O'Donnell (HHMI, Rockefeller University) have shown that yeast MutLα, which contains a DQHA(X)2E(X)4E motif, is also a latent endonuclease and that amino acid substitution mutations within this motif not only inactivate endonuclease activity in vitro but also abolish mismatch repair in the yeast cell. By contrast, E. coli MutL, which lacks a DQHA(X)2E(X)4E motif, is devoid of endonuclease activity. These findings imply that two distinct classes of mismatch repair systems exist in nature: those like the E. coli pathway that rely on GATC methyl-direction and a MutH activity, and those typified by the human and yeast systems that depend on MutL homolog endonuclease activity.
Structure of Human MutSα
A collaborative study with Lorena Beese (Duke University) has yielded the structure of the human mismatch recognition activity MutSα. As in bacterial MutS, mismatch recognition by MutSα occurs via the minor groove of the DNA helix, with a conserved Phe-X-Glu motif of the MSH6 subunit making key contacts with the mispair. As in the bacterial structure, the phenylalanine of this motif stacks upon a mispaired base that hydrogen-bonds to the glutamate residue. These findings have permitted us to visualize in a structural context those amino acid residues that are altered by missense mutations in cancer patients (Figure 2).
Our work on the bacterial and human mismatch repair systems has been supported in part by grants from the National Institutes of Health.
Last updated: October 1, 2007
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