2c7o: Difference between revisions
No edit summary |
No edit summary |
||
| Line 5: | Line 5: | ||
==Overview== | ==Overview== | ||
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 and their structures determined at higher, than 2 A resolution. From time-resolved fluorescence measurements of these, single crystals, we have established that the fluorescence decay function, of AP shows a pronounced, characteristic response to base flipping: the, loss of the very short (approximately 100 ps) decay component and the, large increase in the amplitude of the long (approximately 10 ... | 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 and their structures determined at higher, than 2 A resolution. From time-resolved fluorescence measurements of these, single crystals, we have established that the fluorescence decay function, of AP shows a pronounced, characteristic response to base flipping: the, loss of the very short (approximately 100 ps) decay component and the, large increase in the amplitude of the long (approximately 10 ns), component. When AP is positioned at sites other than the target site, this, response is not seen. Most significantly, we have shown that the same, clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP, fluorescence decay function reveals conformational heterogeneity in the, DNA-enzyme complexes that cannot be discerned from the present X-ray, structures. | ||
==About this Structure== | ==About this Structure== | ||
2C7O is a | 2C7O is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus] with SO4 and SAH as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/DNA_(cytosine-5-)-methyltransferase DNA (cytosine-5-)-methyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.1.37 2.1.1.37] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2C7O OCA]. | ||
==Reference== | ==Reference== | ||
| Line 25: | Line 25: | ||
[[Category: transferase]] | [[Category: transferase]] | ||
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 12:43:27 2007'' | ||
Revision as of 13:38, 5 November 2007
|
HHAI DNA METHYLTRANSFERASE COMPLEX WITH 13MER OLIGONUCLEOTIDE CONTAINING 2-AMINOPURINE ADJACENT TO THE TARGET BASE (PCGC:GMGC) AND SAH
OverviewOverview
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 and their structures determined at higher, than 2 A resolution. From time-resolved fluorescence measurements of these, single crystals, we have established that the fluorescence decay function, of AP shows a pronounced, characteristic response to base flipping: the, loss of the very short (approximately 100 ps) decay component and the, large increase in the amplitude of the long (approximately 10 ns), component. When AP is positioned at sites other than the target site, this, response is not seen. Most significantly, we have shown that the same, clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP, fluorescence decay function reveals conformational heterogeneity in the, DNA-enzyme complexes that cannot be discerned from the present X-ray, structures.
About this StructureAbout this Structure
2C7O is a Protein complex structure of sequences from Haemophilus haemolyticus with SO4 and SAH as ligands. Active as DNA (cytosine-5-)-methyltransferase, with EC number 2.1.1.37 Structure known Active Site: AC1. Full crystallographic information is available from OCA.
ReferenceReference
Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI-DNA complexes., Neely RK, Daujotyte D, Grazulis S, Magennis SW, Dryden DT, Klimasauskas S, Jones AC, Nucleic Acids Res. 2005 Dec 9;33(22):6953-60. Print 2005. PMID:16340006
Page seeded by OCA on Mon Nov 5 12:43:27 2007