The long-term goal of this project is to understand how m5C-cytosine methylases (m5C- methylases) achieve their exquisite specificity in recognizing DNA sequences. To achieve this goal Dr. Roberts is studying a set of enzymes that are related both structurally and functionally. Sequences are available for more than 50 m5C-methylases and recently a structure has been deduced for M.Hhal (recognition sequence: GCGC) both as a binary complex with its cofactor, S-adenosylmethionine, and as a ternary complex with DNA and the end product of the reaction, S- adenosylhomocysteine. From this structure a novel mechanism of DNA binding has been discovered that involves flipping the target cytosine residue completely out of the helix. Future experiments will use site- specific mutagenesis to probe the mechanism of this flipping in great detail, and to explore other aspects of the enzymology of M.Hhal. It is known that DNA recognition is mediated by a variable domain present in each of the m5C-methylases. A series of highly specific domain-swap experiments will be carried out with the idea of using MHhal as a crystallographic framework in which structural information may be obtained about other variable regions to discover if the mechanisms of DNA recognition are similar among this family of enzymes. Additional experiments will test whether base flipping is conserved among other DNA methyltransferases. Finally, protein engineering experiments will be undertaken aimed at modifying M.Hhal so that it can transfer groups other than methyl from the sulphonium center of analogs of AdoMet. These studies will provide information about DNA protein interactions and may help to widen the scope of usefulness of methyltransferases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046127-07
Application #
2392151
Study Section
Biochemistry Study Section (BIO)
Project Start
1991-04-01
Project End
1999-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
New England Biolabs, Inc.
Department
Type
DUNS #
066605403
City
Ipswich
State
MA
Country
United States
Zip Code
01938
Dong, Aiping; Zhou, Lan; Zhang, Xing et al. (2004) Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions. Biol Chem 385:373-9
Pradhan, Sriharsa; Kim, Gun-Do (2002) The retinoblastoma gene product interacts with maintenance human DNA (cytosine-5) methyltransferase and modulates its activity. EMBO J 21:779-88
Bacolla, A; Pradhan, S; Larson, J E et al. (2001) Recombinant human DNA (cytosine-5) methyltransferase. III. Allosteric control, reaction order, and influence of plasmid topology and triplet repeat length on methylation of the fragile X CGG.CCG sequence. J Biol Chem 276:18605-13
Cheng, X; Roberts, R J (2001) AdoMet-dependent methylation, DNA methyltransferases and base flipping. Nucleic Acids Res 29:3784-95
Margot, J B; Aguirre-Arteta, A M; Di Giacco, B V et al. (2000) Structure and function of the mouse DNA methyltransferase gene: Dnmt1 shows a tripartite structure. J Mol Biol 297:293-300
Pradhan, S; Roberts, R J (2000) Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain. EMBO J 19:2103-14
Bacolla, A; Pradhan, S; Roberts, R J et al. (1999) Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated dna. J Biol Chem 274:33011-9
O'Gara, M; Zhang, X; Roberts, R J et al. (1999) Structure of a binary complex of HhaI methyltransferase with S-adenosyl-L-methionine formed in the presence of a short non-specific DNA oligonucleotide. J Mol Biol 287:201-9
Pradhan, S; Bacolla, A; Wells, R D et al. (1999) Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation. J Biol Chem 274:33002-10
Roberts, R J; Cheng, X (1998) Base flipping. Annu Rev Biochem 67:181-98

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