Abstract
The EcoRV DNA methyltransferase introduces a CH3 group at the 6-amino position of the first dA in the duplex sequence d(GATATC). It has previously been reported that the methylase contacts the four phosphates (pNpNpGpA) at, and preceding, the 5'-end of the recognition sequence as well as the single dG in this sequence (Szczelkun, M. D., Jones, H., and Connolly, B. A. (1995) Biochemistry 34, 10734-10743). To study the possible role of the dA and T bases within the ATAT sequence, interference studies have been carried out using diethylpyrocarbonate and osmium tetroxide. The methylase bound very strongly to hemimethylated oligonucleotides modified at the second AT, of the ATAT sequence, in the unmethylated strand of the duplex. This probably arises because these modifications facilitate DNA distortion that follows the binding of the nucleic acid to the protein. Oligonucleotides containing modified bases at both the target dA base and its complementary T were used to determine whether this dA methylase flips out its target base in a similar manner to that observed for dC DNA methylases. In binary EcoRV methylase-DNA complexes, analogues that weakened the base pair caused an increase in affinity between the protein and the nucleic acid. In contrast, in ternary EcoRV methylase-DNA-sinefungin (an analogue of the natural co-factor, S-adenosyl-L-methionine (AdoMet)) complexes, only small differences in affinity were observed between the normal dA-T base pair and the analogues. These results are almost identical to those seen with DNA dC methylases (Klimasauskas, S., and Roberts R. J. (1995) Nucleic Acid Res. 23, 1388-1395; Yang, S. A., Jiang-Cheng, S., Zingg, J. M., Mi, S., and Jones, P. A. (1995) Nucleic Acids Res. 23, 1380-1387) and support a base-flipping mechanism for DNA dA methylases.
Highlights
The EcoRV DNA methyltransferase introduces a CH3 group at the 6-amino position of the first dA in the duplex sequence d(GATATC)
In ternary EcoRV methylase-DNA-sinefungin (an analogue of the natural co-factor, S-adenosyl-L-methionine (AdoMet)) complexes, only small differences in affinity were observed between the normal dA-T base pair and the analogues
The results obtained in this paper are consistent with the EcoRV methylase causing distortions to its GATATC cognate sequence
Summary
(Received for publication, July 9, 1996, and in revised form, September 18, 1996). From the Department of Biochemistry and Genetics, The University of Newcastle, Newcastle upon Tyne, NE2 4HH, United Kingdom. It has been possible to probe DNA structural features such as cruciforms, which show hyper-reactivity [25] In this paper, both DEPC and OsO4 have been used to study the interaction of EcoRV methylase with the ATAT bases within its recognition sequence. Other repair enzymes including uracil-DNA glycosylase [32, 33], the Ada enzyme [34], and Escherichia coli photolyase [35] have had their structures solved, either as apo-enzymes or with short single-stranded oligonucleotides In these cases, the putative distance between the catalytic apparatus and the inferred position of the target base in doublestranded DNA suggested that base flipping might occur. The mismatch base approach has been used with the EcoRV methylase to evaluate base flipping with a DNA dA methylase
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
