Our Scientists

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MLH1 deficiencies were common in the field defects histologically normal tissues surrounding tumors; see Table above. Inverse relationship between microsatellite instability and K-ras and p53 gene alterations in colon cancer. It shows, however, that dam cells are fully proficient at mismatch correction.

In a trans activation model, communication occurs between MutS-MutL complexes at a mismatch and proteins that operate downstream like MutH by protein-protein interactions accompanied by DNA bending. The MMR process is performed by a set of ATPases that transmit, validate, and couple information to identify which DNA strand requires repair.

mismatch repair

Paul Modrich studies pathways that control mutation production. Much of his work has addressed strand-directed mismatch repair. Inactivation of mismatch repair is the cause of one of the most common forms of hereditary cancer, and aberrant action of the pathway plays an important role in the development of a number of neurodegenerative diseases. 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 Lynch syndrome, one of the most common forms of hereditary cancer, and contribute to the development of a subset 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 mistake. Newly synthesized DNA is transiently unmethylated at GATC sequences, and it is the absence of this modification that directs repair to the new strand. The simplest mode of eukaryotic mismatch repair deduced from biochemical studies involves mismatch- and MutSα dependent activation of Exo1, which initiates hydrolysis at a 5' strand break. RPA, which binds to the single-stranded gap produced in this manner, controls the processive action of Exo1 and restricts exonuclease access to the DNA after mismatch removal, resulting in dramatic attenuation of hydrolysis after the mispair has been excised. The RPA-filled gap is repaired by DNA polymerase δ in a reaction that also requires RFC and PCNA. MutLα and PARP-1 are not essential for repair in this simple system, but they substantially enhance mismatch dependence of excision by suppressing Exo1 action on mismatch-free DNA. Because mismatch recognition by MutSα promotes Exo1 initiation at the strand break, signaling must occur between the two DNA sites green arrow. Adapted with modifications from Modrich, P. Journal of Biological Chemistry 281:30305—30309. Figure 2: MutLα dependent modes of mismatch repair. Activation of MutLα endonuclease depends on a mismatch, MutSα PCNA, RFC, ATP, and a DNA strand break. The strand break involved in endonuclease activation may be located either 3' or 5' to the mismatch 3' shown. Incision vertical red arrows is directed to the strand that contains a preexisting break and is biased to the distal side of the mispair to yield an intermediate containing strand breaks to either side of the mismatch. In these multiply incised molecules, 5' termini can serve as entry sites for MutSα-activated Exo1 left pathway , which removes the mismatch by 5' to 3' hydrolysis; the ensuing RPA-filled gap is repaired by DNA polymerase δ with the assistance of PCNA and RFC. The multiply incised intermediate produced by MutLα endonuclease action is also subject to repair by a slower Exo1-independent mechanism right pathway that involves synthesis-driven strand displacement by DNA polymerase δ in the presence of PCNA, RFC, and RPA. Our biochemical studies have shown that 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 one of several single-strand-specific exonucleases. Excision terminates upon mismatch removal, and DNA polymerase III holoenzyme repairs the ensuing gap. Ligase restores covalent continuity to the helix. Correction of DNA Replication Errors in Human Cells In contrast to the E. This permitted us to identify two key activities involved in the initiation of human mismatch repair: MutSα, a heterodimer of the MutS homologs MSH2 and MSH6 that is responsible for most mismatch recognition events in human cells, and MutLα, a heterodimer of the MutL homologs MLH1 and PMS2 that is recruited to the mismatch in a MutSα-dependent manner. These two heterodimers are of interest because genetic inactivation of either causes cancer. This has revealed several reactions that likely contribute to the process of replication error correction in higher cells. RPA, which binds to the single-stranded gap produced in this manner, controls the excision process and down-regulates hydrolysis after mismatch removal. MutLα and PARP-1 are not required for excision in this system; however, by suppressing Exo1 action on mismatch-free DNA they substantially enhance the mismatch dependence of the reaction. The RPA-filled single-stranded DNA gap produced in this manner is repaired by DNA polymerase δ with the assistance of RFC and PCNA. Initiation of this mode of repair involves activation of a latent endonuclease within MutLα in a reaction that requires a mismatch, a DNA strand break, MutSα, RFC, PCNA, and ATP. Strand direction is manifested at this step of repair: action of the MutLα endonuclease is directed to the heteroduplex strand that contains the preexisting break and is biased to the distal side of the mismatch Figure 2, left pathway. These multiply incised molecules are substrates for MutSα-activated Exo1, which removes the mismatch. The PCNA replication clamp plays critical roles in the activation and strand direction of the MutLα endonuclease. The two faces of the PCNA clamp are not equivalent, and several other laboratories, including those of HHMI, Rockefeller University and HHMI, University of California, Berkeley , have shown that PCNA is loaded with a unique orientation at a DNA strand break. We have found that the heteroduplex strand break serves as a site for PCNA loading and that the orientation of clamp loading, relative to the absolute orientation of the helix, determines the strand direction of MutLα incision. These findings also provide a hint concerning the nature of the strand signals that direct mismatch repair in the eukaryotic cell. While Exo1 is believed to play an important role in mammalian mismatch repair, mouse genetic studies indicate that a substantial fraction of repair events can occur in an Exo1-independent manner. We have identified an Exo1-independent mode of mismatch repair that may account, at least in part, for these findings. This reaction, which depends on MutLα endonuclease action, occurs by the mechanism shown in Figure 2 right pathway. After incision by MutLα, the multiply incised heteroduplex serves as substrate for DNA synthesis—driven strand displacement by polymerase δ. This leads to removal of a DNA segment spanning the mismatch and concomitant mismatch repair. Mutagenic Action of Mismatch Repair Although we usually think of mismatch repair as a mutation avoidance system, there are two instances where action of the pathway is required for mutation production. However, because expansion can occur in postmitotic cells, it has been unclear how mismatch repair initiates on nonreplicating DNA. We have identified a simple mechanism by which this may occur. Because these lesions are also recognized by MutSβ, this leads to activation of MutLα endonuclease, which can incise either DNA strand, thus providing an intermediate for downstream repair events. Indeed, covalently continuous heteroduplex DNA containing a 2- or 3-repeat unit extrusion also triggers MutSβ- and MutLα-endonuclease-dependent mismatch repair in nuclear extracts of human cells, and the reaction occurs without obvious strand bias. These findings thus provide a simple mechanism for initiation of triplet repeat processing in nonreplicating DNA. In addition to clarifying the mode of DNA lesion recognition, the MutSα structures have permitted visualization in a structural context of those amino acid residues that are altered by missense mutations in Lynch syndrome patients. Structural features of the Exo1-DNA complex and comparative biochemical analysis of the catalytic domain and full-length forms of the enzyme have also suggested a simple mechanism for allosteric activation of Exo1 by partner proteins like MutSα. Work on the bacterial and human mismatch repair systems has been supported in part by grants from the National Institutes of Health.

Metaplasia is defined as the conversion of a mature differentiated cell into another form of a mature cell type, often following injury or insult. Merck donated the study drug and reviewed the final drafts of the protocol and of this sin before submission; they did not participate in the analysis of the data. An endonuclease then digests the damaged strand past the site of damage. Mismatch repair the quadruple mutant strain has a mutator phenotype and the mutation rate is at least 10-fold lower than expected compared to mutHLS jesus. Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA. A Framework MMR Model Version 3. The 5-meCyt bases are produced by DNA cytosine methyltransferase Dcm at its recognition site -CCAGG- and deamination of 5-meCyt alters it to -CTAGG- Ring 4. No increased mutability was seen with 4-nitroquinoline oxide or ultra-violet irradiation.