DNA methylation is one of numerous mechanisms whereby mammalian gene expression is regulated, and changes in DNA methylation are implicated in mammalian cellular transformation. Methylation patterns are established during gametogenesis and early embryogenesis through the action of DNA (cytosine-5-)-methyltransferase (DNA Mtase); these patterns are maintained by DNA Mtase, enabling the clonal propagation of patterns of gene expression during differentiation. Little is known about what determines the methylation patterns, and in particular, the involvement of DNA Mtase in this process. The recent availability of homogeneous Mtase preparations and the protein sequence provide the opportunity for detailed biochemical investigation of this important enzyme. Using homogeneous DNA Mtase isolated from Friend murine erythroleukemia cells and synthetic DNA substrates we propose to elucidate various aspects of Mtase-DNA interactions. Based on our experience with EcoRI DNA Mtase, we propose to develop a sequence-specific DNA binding assay. This will be used to map the enzyme-substrate interface with DNA- footprinting methods, thus providing detailed information about the size of the interface and whether major and/or minor grooves are contacted. Further, this will allow investigation of what role(s) the large (1000 amino acids) N-terminal domain plays in DNA binding and discrimination of hemi-methylated substrates. Regions of the enzyme involved in DNA and AdoMet recognition will be identified using several cross-linking strategies in combination with mass spectrometric analysis. The binding assay will be used to quantitate enzyme interactions with native, hemi-methylated and single stranded substrates. Comparison of these binding affinities with the corresponding specificity constants (k(cat)/K(m)) should aid our understanding of the enzyme's well known preference for hemi-methylated substrates. We will determine if the recently reported substrate inhibition occurs as a result of complex enzyme-enzyme interactions or through multiple DNA substrates binding the same enzyme molecule. The reported inhibition of enzyme activity deriving from """"""""nonsubstrate nucleic acids"""""""" will be mechanistically investigated because of the possible regulatory importance. Results from the proposed experiments will be used in future studies of sequence- specificity and isolation of cellular factors which modulate Mtase activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046333-03
Application #
3305749
Study Section
Biochemistry Study Section (BIO)
Project Start
1991-09-25
Project End
1995-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Santa Barbara
Department
Type
Schools of Arts and Sciences
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
Estabrook, R August; Lipson, Rebecca; Hopkins, Ben et al. (2004) The coupling of tight DNA binding and base flipping: identification of a conserved structural motif in base flipping enzymes. J Biol Chem 279:31419-28
Malygin, Ernst G; Lindstrom Jr, William M; Zinoviev, Victor V et al. (2003) Bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase): evidence for two distinct stages of methylation under single turnover conditions. J Biol Chem 278:41749-55
Glickman, J F; Flynn, J; Reich, N O (1997) Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase. Biochem Biophys Res Commun 230:280-4
Flynn, J; Glickman, J F; Reich, N O (1996) Murine DNA cytosine-C5 methyltransferase: pre-steady- and steady-state kinetic analysis with regulatory DNA sequences. Biochemistry 35:7308-15
Xu, G; Flynn, J; Glickman, J F et al. (1995) Purification and stabilization of mouse DNA methyltransferase. Biochem Biophys Res Commun 207:544-51