The Investigation of DNA-Methyltransferase Interactions in the Adenine Methyltransferases using the Time-resolved Fluorescence of 2-Aminopurine
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2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.
DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a ba...
متن کاملUnusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase.
The methyltransferase, M.EcoKI, recognizes the DNA sequence 5'-AACNNNNNNGTGC-3' and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-aminopurine is often used as a signal for base flippin...
متن کاملUsing the fluorescence decay of 2-aminopurine to investigate conformational change in the recognition sequence of the EcoRV DNA-(adenine-N6)-methyltransferase on enzyme binding.
The EcoRV DNA methyltransferase methylates the first adenine in the GATATC recognition sequence. It is presumed that methylation proceeds by a nucleotide flipping mechanism but no crystal structure is available to confirm this. A popular solution-phase assay for nucleotide flipping employs the fluorescent adenine analogue, 2-aminopurine (2AP), substituted at the methylation target site; a subst...
متن کاملTime-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes
DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared ...
متن کاملSubstrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase.
RSR:I [N:6-adenine] DNA methyltransferase (M.RSR:I), which recognizes GAATTC and is a member of a restriction-modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M.RSR:I were performed on unmethylated, hemimethylated, dimethylated or no...
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تاریخ انتشار 2008