MutS DNA Mistmatch Repair Protein
Eric Stoffregen and
CLU Biology Department
©David Marcey, 2001
II. Protein Structural Features
III. DNA Mismatch Features
IV. Protein-DNA Interactions
V. Dimer Interactions
VI. Nucleotide Binding and Hydrolosis
Note: This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.
The MutS protein of Escherichia coli, shown on the left complexed to a DNA substrate with a mismatch G-T, is responsible for repairing errors in DNA replication. MutS is a mismatch repair (MMR) protein that increases the fidelity of DNA replication 100-1,000 times. MutS, with the help of two other MMR proteins, MutH and MutL, recognizes and repairs numerous errors, including mismatches, unpaired bases, and small insertion or deletion loops.
Figure 1 depicts the role of MutH, MutS, and MutL in methyl-directed mismatch repair. Once MutS recognizes an error and associates with MutL, the complex in turn activates the latent endonuclease activity of MutH. MutH cleaves on one side of the mismatch at a hemi-methylated (GATC) sequence. Depending on whether MutH cuts on the 5' or 3' side of the mismatch, either exonuclease VII or exonuclease I is employed (together with MutS, MutL, and helicase II) to remove a stretch of DNA between the MutH cut and the mismatch. Repair synthesis followed by ligation restores a wild type dsDNA sequence. Please reload the molecule before proceeding.
Homologues of this E. coli MutS protein are found in almost every organism. In prokaryotes, MutS proteins originate from a single gene, while eukaryotes (humans included) contain multiple mutS homologue (msh) genes. Mutations in human MutS homologues have been linked to heritable forms of cancer, indicating the importance of this protein in DNA repair.
MutS is a large protein (125 x 90 x 55 Å) composed of numerous alpha helices and beta sheets . MutS is homodimeric, and the two identical monomers differ only in the relative orientations of the mismatch-binding and non-mismatch-binding monomers . Each monomer of the MutS protein has several domains, each resembling at least one previously determined structure. The helix-turn-helix (HTH) domain is involved in dimer contacts . The ATPase domain, responsible for the binding and hydrolysis of ATP most closely resembles the ATPase domain found in the ABC ATPase superfamily . The connector domain, which connects the mismatch domain to the core domain, is a mostly parallel beta sheet with four surrounding alpha helices . The structure of the connector domain resembles the Holliday junction resolvase. The mismatch domain contains a six-stranded beta sheet three surrounding alpha helices . The mismatch domain has similar structure to a transfer RNA endonuclease. The core domain has two regions that from a helical bundle, with two additional alpha helices extending as levers toward the DNA . Finally, the clamp domain, involved in DNA recognition and binding, is characterized by a four stranded antiparallel beta sheet . The DNA backbone fits into the top of the dimer between the clamp domains and the mismatch domains . This view shows only the mismatch-binding monomer with the DNA.
A full picture of how the protein and DNA fit together is achieved by viewing the way each domain in the mismatch-recognition monomer fits together with each corresponding domain in the non-mismatch-binding monomer (HTH, ATPase, connector, mismatch, core, and clamp domains) . An even better model of how the DNA fits into the protein can be seen by viewing a spacefill representation of the protein. Once again, the domains of the protein will be colored as above .
The mismatch in this DNA is the mispairing of a guanine (G9) base with a thymine (T22) base, instead of G-C or A-T, causing distortions in the orientations of the bases . The G-T mismatch does not form as a wobble base pair, but rather shows a very different hydrogen bonding pattern. In wobble pairing, the bases would have hydrogen bonds between guanine 06 and thymine N3, but these bases have hydrogen bonding between N1 and N2 of guanine 9 (G9) and O4 of thymine 22 (T22) .
The mismatch bases remain in the DNA helix, rather than being flipped out. The bases at the mismatch do not stack, and as a result a kink of ~60° occurs in this region. The DNA adapts to the kink by adjusting the puckering of the sugars in the DNA from the B DNA form (C2'-endo) to the A DNA form (C3'-endo) for six of the nucleotides surrounding the mismatch. The combination of the change in sugar puckering, the unusual hydrogen bonding between the mismatched bases, and the protein pulling on the DNA at the mismatch site results in changes to the major and minor groove of the DNA at the mismatch region. For base pairs 8-11, the DNA has a very wide and shallow minor groove, and a narrow and deep major groove .
Only the mismatch-recognition monomer has specific contacts to the mismatch DNA. The non-mismatch binding monomer only has loose DNA backbone contacts.
Initial DNA recognition is the responsibility of the clamp domains. The clamp domains have limited, sequence-independent contacts with the DNA backbone. The clamp domain of the non-mismatch binding monomer spans the deep major groove of the DNA mismatch region, while the clamp domain of the mismatch-binding monomer sits slightly to the side .
The clamp domains are the only part of the MutS protein with a largely positively charged surface. The clamp domains present this positively charged surface to the DNA, forming salt bridges with the DNA phosphates (allow time to load). Positive, neutral, and negative electrostatic potentials on the protein surface are indicated by blue, white, and red, respectively.
The mismatch-binding domain of the mismatch-recognition monomer contacts the DNA on the minor groove side. Three major interactions occur between the DNA and protein that allow the recognition of the mismatched bases. The amino acid Glu 38 forms hydrogen bonds to guanine 10, and to thymine 22 of the mismatch . Asp 35 forms a hydrogen bond with guanine 9 of the mismatch .
Ordinarily, the negatively charged side chains of these two amino acids (Glu 38 and Asp 35) would cause electrostatic repulsion of the DNA backbone, but since the minor groove is widened, the side chains can sit in the middle of the minor groove with no difficulties. The third interaction is the wedging of Phe 36 into the DNA, which stacks with the T22 mismatch . It appears that those three amino acids are already oriented as MutS probes the DNA replication errors. The phenylalanine (Phe) will only wedge in where the DNA backbone is flexible due to a mismatch or looped out base.
V. Dimer Interactions
Interactions between the two monomers of MutS occur in the clamp domains and the ATPase/HTH region. The clamp domains interact by sharing a small contact surface area and two intramolecular hydrogen bonds between the non-mismatch-binding monomer and the mismatch-binding monomer .
The interactions between the ATPase/HTH regions are much more extensive. The two alpha helices of the mismatch-binding monomer's HTH domain bundle with two alpha helices of the non-mismatch-binding monomer's ATPase domain. The two alpha helices of the non-mismatch-binding monomer's HTH domain pair up in the same manner with two alpha helices of the ATPase domain of the mismatch-binding monomer . These regions of the dimer are mostly hydrophobic, as the next button will illustrate by highlighting the hydrophobic residues . The interface between the proteins is 2,922 Å2, which, as you can see from this spacefill representation of the region, is a huge amount of contact surface .
VI. Nucleotide Binding and Hydrolysis
ADP and Mg2+ are found bound to the mismatch-binding monomer only, which fits with data indicating that the two ATPase domains are not identical. The mismatch-binding monomer binds ADP in a P-loop fashion. The adenine group of ADP stacks between two amino acids of the ATPase domain, His 760 and Phe 596 . Specificity for the ADP molecule is then achieved by the formation of two hydrogen bonds between ADP and Ile 597 . Further stability of ADP-binding occurs through the octahedral coordination of the Mg2+ ion to the B phosphate of the ADP molecule, the hydroxyl group of Ser 621, and four water molecules . Four residues from a Walker B motif that stabilizes the waters bound to the Mg2+ ..
The non-mismatch-binding monomer will most likely continue with ATP uptake, which may then cause the release of MutS from the mismatch site. These results, however, may not be accurate, as they were found in the absence of MutL, which may prevent MutS from leaving the mismatch even in the presence of ATP. Previous models have indicated that the MutS protein leaves the mismatch in one of two ways. It may leave via ATP-hydrolysis dependent translocation, or it may slide down the DNA prior to the mismatch repair. Further research is needed to determine what exactly the function of the ATPase activity of MutS is, although the necessity and importance of this aspect is not disputed.
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Lamers, M.H., Perrakis, A., Enzlin, J.H., Winterwerp, H.H.K., de Wind, N., Sixma, T.K (2000). The crystal structure of DNA mismatch repair protein MutS binding to a G-T mismatch. Nature. 407: 711-717.
Obmolova, G., Ban, C., Hsieh,
P., Yang, W. (2000). Crystal structures of mismatch repair protein MutS and
its complex with a substrate DNA. Nature. 407: 703-710.