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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 repair system has profound consequences for a living cell: a 100- to 1,000-fold increase in mutation production. In humans, mismatch repair defects are the cause of a common form of hereditary colon cancer (HNPCC) but are also believed to play a role in the development of 15–25 percent of sporadic tumors that occur in a variety of tissues.
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, which corrects errors that occur 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 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.
Correction of DNA Replication Errors in Human Cells
DNA methylation does not play a role in strand direction of eukaryotic mismatch repair, and in contrast to E. coli, the eukaryotic organisms examined to date do not possess a MutH activity. Although the strand signals that direct replication error correction in eukaryotic cells have not been genetically identified, a DNA strand break is sufficient to direct mismatch repair in human cell extracts. The strand break that directs the reaction may reside 3' or 5' to the mismatch at a distance of as much as 1,000 base pairs. The key proteins responsible for initiation of human mismatch repair are MutSα (a heterodimer of the MutS homologs MSH2 and MSH6) and MutLα (a heterodimer of MutL homologs MLH1 and PMS2). MutSα, which is responsible for most mismatch recognition events in human cells, recruits MutLα 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.
To study the mechanism by which replication error correction occurs in human cells, we have reconstituted 3'- and 5'-directed mismatch repair, using a set of purified 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). This set of proteins is sufficient to support strand-directed mismatch correction, and analysis of this purified system has clarified basic features of 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 a DNA strand break. Strand direction is manifested at this step of repair: 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. These multiply incised molecules serve as substrates for MutSα-activated Exo1, which loads at a 5' strand break and removes the mismatch by 5' to 3' hydrolysis. The resulting 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 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 the existence of two distinct classes of mismatch repair system in nature: those like the E. coli pathway that rely on GATC methyl-direction and a MutH activity, and those exemplified by the human and yeast systems that depend on MutL homolog endonuclease activity.
As mentioned above, activation and strand direction of the MutLα endonuclease depends on a mismatch, MutSα, RFC, PCNA, and a DNA strand break. Analysis of this reaction has demonstrated that RFC function in MutLα activation is restricted to loading of the PCNA clamp onto the helix. The two faces of the PCNA clamp are not equivalent, and several other laboratories, including those of Michael O'Donnell (HHMI, Rockefeller University) and John Kuriyan (HHMI, University of California, Berkeley), have shown that PCNA is loaded with a unique orientation at a DNA strand break. This finding, coupled with the fact that the MutLα forms a 1:1 complex with the PCNA clamp, has led us to suggest that the orientation of clamp loading dictates the strand direction of DNA incision by MutLα. We have also found, consistent with this view, that a short region of unpaired helix can substitute for a strand break as a site of PCNA loading. PCNA loaded onto such a bubble-containing heteroduplex supports MuLα activation, but strand direction of endonuclease incision is lost. As illustrated in Figure 2, this is the expected result if the orientation of clamp loading determines strand direction of MutLα action. Because PCNA is a key component of the DNA replication apparatus at the replication fork, these findings suggest that DNA termini that direct clamp loading at the fork may serve as the strand signals that direct mismatch repair in the eukaryotic cell.
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 3. 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.
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 29, 2010
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