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Enzymatic capture of an extrahelical thymine in the search for uracil in DNA

Department of Pharmacology and Molecular Sciences, Johns Hopkins Medical School, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Nature (Impact Factor: 42.35). 10/2007; 449(7161):433-7. DOI: 10.1038/nature06131
Source: PubMed

ABSTRACT The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

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    • "However, recent work has shown that MutM may promote lesion extrusion by inducing a bend in the DNA (Qi et al., 2009). Unlike UNG, which passively captures spontaneously extruded lesions (Parker et al., 2007), hOgg1 can dramatically bend DNA like MutM, even in the absence of a lesion, as has been directly visualized by a single-molecule imaging method (Chen et al., 2002). For hOgg1, the enzyme binding energy may strain the DNA and assist in base flipping (Friedman and Stivers, 2010); however , there is a lack of direct evidence for this mechanism. "
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    • "into the catalytic pocket of the enzyme ( Parker et al . , 2007 ; Slupphaug et al . , 1996 ) . Thus , DG ' s specificity results from the fit of the substrate lesion into the binding pocket where the binding is stabilized by various types of interactions . However , because a number of oxidized bases ( >20 ) are repaired by only four ( or five ) DGs in the mammalian cells , the DGs usually possess b"
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    • "Here we have investigated whether obstacles of another nature, where double-stranded DNA is interrupted by a single-stranded stretch, affect the processive search by Ung. Although no structure of the complex of E. coli Ung with any DNA is available, the human homolog forms contacts with both strands when searching undamaged double-stranded DNA [25], so it is reasonable to suggest that the protein must undergo a conformational change when transferred from double-to single-stranded DNA, or release DNA and re-associate with it in another binding mode. We have constructed double-stranded substrates containing 2-, 4-, or 6-nt long gaps in the non-damaged strand, or containing a nick (essentially, a one-phosphate gap), and compared the correlated cleavage of such substrates by Ung (Fig. 3). "
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