Nucleic Acids Research, 2000, Vol. 28, No. 9 1906-1912
© 2000 Oxford University Press
Structural basis for uracil DNA glycosylase interaction with uracil: NMR study
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India and 1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop. The 1H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin. This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T12 and T15 nucleotides stack upon 3' and 5' ends of the stem, respectively. Further, T14 stacks upon both T12 and T15. Though U13 partially stacks upon T14, no stacking interaction is observed between U13 and T12. All the individual nucleotide bases belonging to the stem and T12 and T15 of the loop adopt ‘anti’ conformation with respect to their sugar moiety, while the U13 and T14 of the loop are in ‘syn’ conformation. The turning phosphate in the loop is located between T13 and T14. This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. The 
of T12 and 
, ß, 
, and 
of U13 and of T14, which partially influence the local conformation of U13 in U2-hairpin are all locked in ‘trans’ conformation. Such stretched out backbone conformation in the vicinity of U13 could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases.
* To whom correspondence should be addressed. Tel: +91 22 215 2971; Fax: +91 22 215 2110; Email: chary@tifr.res.in
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. J. Wilton, M. Ghosh, K. V. A. Chary, K. Akasaka, and M. P. Williamson Structural change in a B-DNA helix with hydrostatic pressure Nucleic Acids Res., July 1, 2008; 36(12): 4032 - 4037. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R.W. Bellamy, K. Krusong, and G. S. Baldwin A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping Nucleic Acids Res., March 12, 2007; 35(5): 1478 - 1487. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Handa, S. Roy, and U. Varshney The Role of Leucine 191 of Escherichia coli Uracil DNA Glycosylase in the Formation of a Highly Stable Complex with the Substrate Mimic, Ugi, and in Uracil Excision from the Synthetic Substrates J. Biol. Chem., May 11, 2001; 276(20): 17324 - 17331. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Handa, N. Acharya, and U. Varshney Chimeras between Single-stranded DNA-binding Proteins from Escherichia coli and Mycobacterium tuberculosis Reveal That Their C-terminal Domains Interact with Uracil DNA Glycosylases J. Biol. Chem., May 11, 2001; 276(20): 16992 - 16997. [Abstract] [Full Text] [PDF]
|
|