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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 mutation production. Inactivation of the human mismatch repair system and the ensuing genetic instability are 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 adaptation 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.
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
DNA methylation is not believed to play a role in eukaryotic mismatch repair, in contrast to mismatch repair in E. coli, 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 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 this purified system has clarified the mechanism of human mismatch repair and has identified a novel function of MutLα that accounts for its involvement in the reaction.
Initiation of human mismatch repair involves activation of a latent endonuclease of MutLα in a reaction that requires a mismatch, MutSα, RFC, PCNA and ATP. Action of this endonuclease is directed to the heteroduplex strand that contains a preexisting break and is biased to the distal side of the mismatch (Figure 1). Incision in this manner yields molecules that contain strand breaks to either side of the mismatch, and these serve as substrates for mismatch-provoked excision. As implied above, Exo1 is the only excision activity that has been implicated in eukaryotic mismatch repair. MutSα activates Exo1 in a mismatch-dependent manner, with the activated exonuclease loading at a 5' strand break in the multiply incised product that results from MutLα action; Exo1 hydrolysis leads to removal of DNA spanning the mismatch; and the ensuing gap is filled by RPA and repaired by DNA polymerase δ in a reaction that also depends on PCNA and RFC.
The latent endonuclease activities of human and yeast MutLα depend on the integrity of a DQHA(X)2E(X)4E metal-binding site located near the C terminus of the PMS2 subunit. This amino acid sequence element is conserved in PMS2 homologs and in many archaeal and eubacterial MutL proteins. However, it is not found in the MLH1 subunit of MutLα and is also conspicuously absent in MutL proteins from bacteria such as E. coli that rely on GATC methylation to direct mismatch repair. Amino acid substitution mutations within the DQHA(X)2E(X)4E metal-binding motif abolish MutLα endonuclease activity in vitro and mismatch repair in the eukaryotic 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.
Although Exo1 is the primary excision activity in mammalian mismatch repair, genetic studies in mice indicate that about 30 percent of repair events can occur in an Exo1-independent manner, an effect that is of interest because Exo1-deficient mice are less prone to cancer than are animals lacking MutSα or MutLα. A possible explanation for this finding was provided by analysis of a purified set of components consisting of MutSα, MutLα, RFC, PCNA, RPA, and DNA polymerase δ. Mismatch repair by this system is less efficient than that which occurs when these six components are supplemented with Exo1, but the reduced rate of repair is nevertheless sufficient to account for Exo1-independent repair events that have been observed in mouse cells. Study of the Exo1-independent reaction has demonstrated that it occurs by the mechanism illustrated in Figure 2. Repair is initiated by endonucleolytic incision of the nicked heteroduplex strand by MutLα. The multiply incised heteroduplex then serves as substrate for DNA synthesis–driven strand displacement by polymerase δ. This results in removal of a DNA segment spanning the mismatch and concomitant mismatch repair.
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 in 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 3).
Work on the bacterial and human mismatch repair systems has been supported in part by grants from the National Institutes of Health.
Last updated September 09, 2009
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