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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Available from: Mario Antonio Bianchet, Aug 09, 2015
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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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    ABSTRACT: 7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative DNA lesions. 8-oxoguanine DNA glycosylases (Oggs) detect and excise 8-oxoG through a multiple-step process. To better understand the basis for estranged base recognition, we have solved the crystal structures of MBOgg1, the 8-oxoguanine DNA glycosylase of Thermoanaerobacter tengcongensis, in complex with DNA containing a tetrahydrofuranyl site (THF, a stable abasic site analogue) paired with an estranged cytosine (MBOgg1/DNA(THF:C)) or thymine (MBOgg1/DNA(THF:T)). Different states of THF (extrahelical or intrahelical) are observed in the two complexes of the ASU of MBOgg1/DNA(THF:C) structure. Analyses of their different interaction modes reveal that variable contacts on the 5' region flanking the THF abasic site are correlated with the states of the THF. Comparison of MBOgg1/DNA(THF:T) with MBOgg1/DNA(THF:C) indicates that the non-preferred estranged T may affect MBOgg1's contacts with the 5' flank of the lesion strand. Furthermore, we identified a region in MBOgg1 that is rich in positive charges and interacts with the 5' region flanking the lesion. This region is conserved only in non-eukaryotic Oggs, and additional mutagenesis and biochemical assays reveal that it may contribute to the distinct estranged base specificities between eukaryotic and non-eukaryotic Oggs.
    Journal of Structural Biology 12/2012; 181(3). DOI:10.1016/j.jsb.2012.12.003 · 3.23 Impact Factor
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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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    ABSTRACT: Oxidative genome damage induced by reactive oxygen species includes oxidized bases, abasic (AP) sites, and single-strand breaks, all of which are repaired via the evolutionarily conserved base excision repair/single-strand break repair (BER/SSBR) pathway. BER/SSBR in mammalian cells is complex, with preferred and backup sub-pathways, and is linked to genome replication and transcription. The early BER/SSBR enzymes, namely, DNA glycosylases (DGs) and the end-processing proteins such as abasic endonuclease 1 (APE1), form complexes with downstream repair (and other noncanonical) proteins via pairwise interactions. Furthermore, a unique feature of mammalian early BER/SSBR enzymes is the presence of a disordered terminal extension that is absent in their Escherichia coli prototypes. These nonconserved segments usually contain organelle-targeting signals, common interaction interfaces, and sites of posttranslational modifications that may be involved in regulating their repair function including lesion scanning. Finally, the linkage of BER/SSBR deficiency to cancer, aging, and human neurodegenerative diseases, and therapeutic targeting of BER/SSBR are discussed.
    Progress in molecular biology and translational science 01/2012; 110:123-53. DOI:10.1016/B978-0-12-387665-2.00006-7 · 3.11 Impact Factor
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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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    ABSTRACT: Uracil-DNA glycosylase (Ung) is a DNA repair enzyme that excises uracil bases from DNA, where they appear through deamination of cytosine or incorporation from a cellular dUTP pool. DNA repair enzymes often use one-dimensional diffusion along DNA to accelerate target search; however, this mechanism remains poorly investigated mechanistically. We used oligonucleotide substrates containing two uracil residues in defined positions to characterize one-dimensional search of DNA by Escherichia coli Ung. Mg(2+) ions suppressed the search in double-stranded DNA to a higher extent than K(+) likely due to tight binding of Mg(2+) to DNA phosphates. Ung was able to efficiently overcome short single-stranded gaps within double-stranded DNA. Varying the distance between the lesions and fitting the data to a theoretical model of DNA random walk, we estimated the characteristic one-dimensional search distance of ~100 nucleotides and translocation rate constant of ~2×10(6) s(-1).
    Biochemical and Biophysical Research Communications 10/2011; 414(2):425-30. DOI:10.1016/j.bbrc.2011.09.106 · 2.28 Impact Factor
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