1H, 13C, and 15N Resonance Assignments of C-terminal Domain of MutY

* References for biological significance of the protein:
-Noll, D.M., Gogos, A., Granek, J.A., & Clarke, N.D.,
(1999) Biochemistry, 38, 6374-6379.
-Guan, Y., Manuel, R.C., Arvai, A.S., Parikh, S.S.,
Mol, C.D., Miller, J.H., & Lloyd, R.S.,
(1998) Nat. Struct. Biol, 5, 1058-1064

* References for the NMR experiment
-Muhandiram, D.R., & Kay, L.E., (1994) J. Magn. Reson. B.,
103, 203-216

The MutY protein, of which the p13_c-terminal_domain is a fragment, functions as a DNA glycosylase (Adenine glycosylase)

Volk, D. E., Thiviyanathan, V., House, P. G., Lloyd, S., and Gorenstein, D. G.,
"1H, 13C, and 15N Resonance Assignments of C-terminal Domain
of MutY," J. Biomol. NMR, submitted.

1H, 13C and 15N resonance assignments of the C-terminal domain of MutY: an adenine glycosylase active on G:A mismatches.

Proteolysis of MutY with trypsin indicated that this DNA mismatch repair enzyme
could exist as two modules and that the N-terminal domain (Met1-Lys225),
designated as p26, could serve as the catalytic domain [Manuel et al. (1996) J.
Biol. Chem. 271, 16218-16226]. In this study, the p26 domain has been cloned,
overproduced, and purified to homogeneity. Synthetic DNA duplexes containing
mismatches, generated with regular bases and nucleotide analogs containing
altered functional groups, have been used to characterize the substrate
specificity and mismatch repair efficiency of p26. In general, p26 recognized
and cleaved most of the substrates which were catalyzed by the intact protein.
However, p26 displayed enhanced specificity for DNA containing an inosine.
guanine mismatch, and the specificity constant (Kcat/Km) was 2-fold higher. The
truncated MutY was able to cleave DNA containing an abasic site with equal
efficiency. Dissociation constants (Kd) were obtained for p26 on noncleavable
DNA substrates containing a tetrahydrofuran (abasic site analog) or a reduced
abasic site. p26 bound these substrates with high specificity, and the Kd
values were 3-fold higher when compared to the intact MutY. p26 contains both
DNA glycosylase and AP lyase activities, and we provide evidence for a reaction
mechanism that proceeds through an imino intermediate. Thus, we have shown for
the first time that deletion of 125 amino acids at the C-terminus of MutY
generates a stable catalytic domain which retains the functional identity of
the intact protein.

Cloning, overexpression, and biochemical characterization of the catalytic domain of MutY.

adenine glycosylase [Escherichia coli O157:H7 str. Sakai]

adenine DNA glycosylase [Escherichia coli str. K12 substr. W3110]

adenine DNA glycosylase [Escherichia coli O111:H-]

adenine glycosylase [Escherichia coli SE11]

adenine DNA glycosylase MutY [Escherichia coli O26:H11 str. 11368]

unnamed protein product [Escherichia coli K-12]

A/G-specific adenine glycosylase [Escherichia coli LF82]

adenine glycosylase; G.C--> T.A transversions [Escherichia coli BL21(DE3)]

adenine DNA glycosylase [Escherichia fergusonii ATCC 35469]

adenine DNA glycosylase [Escherichia coli IAI1]

CG Site No. 18130; alternate name micA [Escherichia coli str. K-12 substr. MG1655]

A/G-specific adenine glycosylase [Escherichia coli]

adenine DNA glycosylase [Escherichia coli str. K-12 substr. MG1655]

adenine glycosylase; G.C --> T.A transversions [Escherichia coli O157:H7 str. EDL933]

adenine glycosylase [Shigella flexneri 2a str. 301]

adenine DNA glycosylase [Escherichia coli O157:H7 str. Sakai]

adenine DNA glycosylase [Shigella flexneri 2a str. 301]

MULTISPECIES: A/G-specific adenine glycosylase [Enterobacteriaceae]

RecName: Full=A/G-specific adenine glycosylase