Jessica Carney (1),
Nathan Silva (3)
and David Marcey (2)
© David Marcey 2001
II. MutH Structure
III. Activation of MutH
IV. Similarity to Restriction Endonucleases
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.
Despite the remarkable fidelity with which DNA polymerases replicate DNA, errors occur with measurable frequencies. Although some errors lead to mutations that increase an organism's fitness, most mutations are deleterious. Selection has thus evolved complex systems to surveil the genome for mutations and DNA damage and to repair these defects. The MutH protein of Escherichia coli, a weak endonuclease, is one enzyme of a multimeric complex that works to repair base mismatches (with the exception of C-C pairs) and small insertion or deletion mismatches in strands differing in up to four nucleotides.
Figure 1 depicts the role of MutH, MutS, and MutL in methyl-directed mismatch repair. Once MutS, another mismatch repair protein, 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 molecule before proceeding to the next section.
The MutH protein (229 residues, 28 kD) is a clamp shaped molecule defined by a large cleft that separates two major subdomains. The N subdomain comprises residues 1-83 and 120-145, forming helices a A, a B, a C, and a mixed b sheet . The C subdomain comprises residues 90-117 and 148-229, forming helices a D, a E, a F, an anti-parallel b sheet, and a b hairpin at the terminus . The two subdomains meet at a region of hydrophobic residues and are connected by three polypeptide linkers . This linkage at this interface provides flexibility, allowing the two subdomains to pivot with respect to each other.
The active site of MutH is located in the cleft located between the N-arm and the C-arm of the molecule. The cleft, similar to that of many restriction endonucleases, is 15-18 angstroms wide and 12-14 angstroms deep. The DNA-binding cleft contacts 7 base pairs of B-DNA . Three residues in the DNA-binding cleft, Asp70, Glu77, and Lys 79, are critical for endonuclease cleavage . These residues form a catalytic triad of the form D(X)6-30(E/D)XK. Such triads are known to be important in the catalytic activity of several type II restriction enzymes (see below).
In addition, two other residues in the binding cleft, Asp91 and Phe94, are highly conserved . Although the function of Asp91 is unclear, Phe94 is known to be entirely exposed to solvent in the free enzyme. It probably aids in DNA recognition or holds the DNA in place by means of intercalation between the substrate base pairs.
Magnesium ions are required for MutH cleavage of targeted DNA strands. The interaction of MutH with Mg2+ ions is likely similar to that of the restriction endonuclease EcoRV, to which it shows structural homology . Glu45 of EcoRV has been shown to coordinate a Mg2+ ion that is critical in catalysis . The analagous residue in MutH is Glu56, which forms a water-mediated hydrogen bond with Glu77 .
MutH must be activated by MutS and MutL to cleave DNA. The exact mechanism of activation is not known, but it appears that the C-arm pivots with respect to the N-arm to open and close the DNA-binding cleft. This turns on and off catalytic activity. The pivoting motion seems to involve the C-terminal a F helix . It is likely that this helix serves as a sort of molecular button which, when "pressed" by MutS and MutL, activates the latent endonuclease activity of MutH.
MutH has been crystallized in two conformations . The first structure, the likely conformation of the active enzyme, displays a closed cleft and has a a F helix that is packed tightly into the structure . The second structure, likely the inactive form of the enzyme, has a more open conformation and an a F helix that protrudes into solution . The difference in a F helix packing suggests that this helix does indeed serve as a lever that is acted upon by MutS and MutL.
MutH activity is similar to that of restriction endonucleases and it is thus intersting that it has considerable sequence homology to Sau3AI and structural similarity to PvuII. Additionally, as discussed above, the catalytic triad found in MutH is of the form D(X)6-30(E/D)XK. This is a motif found in the active sites of many restriction endonucleases, including Eco RI , PvuII , Eco RV , Fok I , and Bam HI .
There are considerable differences between these enzymes. For example, some are dimers and some are monomers and they exhibit specificity for different DNA sequences. Nevertheless, the presence of a common motif suggests that they may be evolutionary homologs, i.e. descended from a common ancestral protein. The ability of bacteria to defend themselves against viral attack by DNA restriction may have evolved from an essential function of DNA repair.
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