Computational Approaches for Investigating Base Flipping in Oligonucleotides

Priyakumar, U. Deva; MacKerell, Alexander D.

doi:10.1021/cr040475z

Cited by 87 publications

(131 citation statements)

References 130 publications

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“…To examine the role of Ala145 on nucleotide flipping for TDG, we performed molecular dynamics (MD) simulations, an approach used previously to study spontaneous and enzyme-mediated nucleotide flipping (45)(46)(47). The MD simulations were initiated from the TDG-G·U F structure, modified as needed to replace the U F analog with deoxythymidine (dT) or dU, or generate TDG variants.…”

Section: Resultsmentioning

confidence: 99%

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

Maiti

Noon

MacKerell

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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168

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DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease. D NA base excision repair (BER) plays an established role in maintaining genomic integrity, and recent studies indicate that BER is also essential for active DNA demethylation, a key element of epigenetic gene regulation (1-3). A central player in both processes is thymine DNA glycosylase (TDG), which initiates BER by excising damaged or modified forms of 5-methylcytosine (mC) that arise at 5′-CpG-3′ sites. TDG was discovered for its ability to selectively remove T from G·T mispairs, mutagenic lesions that can arise from deamination of mC to T (4, 5). TDG also excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), oxidized forms of mC that are generated by Tet enzymes (6, 7). In addition, TDG was shown to be essential for active DNA demethylation and for embryonic development (8, 9), a role that likely involves excision of deaminated or oxidized forms of mC generated by a deaminase or Tet enzyme (7,(10)(11)(12). Thus, the ability of TDG to remove deaminated and oxidized forms of mC is important for genetic and epigenetic integrity.The question of how DNA glycosylases remove modified bases without acting upon the huge excess of undamaged DNA remains largely unresolved, particularly for mismatch glycosylases such as TDG and MBD4 (methyl binding domain IV) (13-15). These enzymes face the formidable task of removing a normal base, t...

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Section: Resultsmentioning

confidence: 99%

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

Maiti

Noon

MacKerell

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

168

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“…Lebrun and Lavery (16) proposed that the 5Ј5Ј structure would have base pairs pulled outwards or flipped whereas the 3Ј3Ј structure would not. To test this prediction, we examined the hysteresis in the presence of ␤-cyclodextrin, a macrocycle that reacts specifically with DNA bases that are extruded from the helix to form a host-inclusion compound (22,23). We find that, for DNA overstretched from the 5Ј5Ј ends in 150 mM NaCl, the difference between the hysteresis in cyclodextrin and the hysteresis without was 2.8 times the standard deviation for hysteresis in the absence of cyclodextrin (above).…”

Section: Hysteresis Of 3 3 and 5 5 Structures Are Differentially Affementioning

confidence: 99%

The structure of DNA overstretched from the 5′5′ ends differs from the structure of DNA overstretched from the 3′3′ ends

Danilowicz¹,

Limouse²,

Hatch³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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It has been suggested that the structure that results when doublestranded DNA (dsDNA) is pulled from the 3 3 ends differs from that which results when it is pulled from the 5 5 ends. In this work, we demonstrate, using phage dsDNA, that the overstretched states do indeed show different properties, suggesting that they correspond to different structures. For 3 3 pulling versus 5 5 pulling, the following differences are observed: (i) the forces at which half of the molecules in the ensemble have made a complete force-induced transition to single stranded DNA are 141 ؎ 3 pN and 122 ؎ 4 pN, respectively; (ii) the extension vs. force curve for overstretched DNA has a marked change in slope at 127 ؎ 3 pN for 3 3 and 110 ؎ 3 pN for 5 5 ; (iii) the hysteresis (H) in the extension vs. force curves at 150 mM NaCl is 0.3 ؎ 0.8 pN m for 3 3 versus 13 ؎ 8 pN for 5 5 ; and (iv) 3 3 and 5 5 molecules show different changes in hysteresis due to interactions with ␤-cyclodextrin, a molecule that is known to form stable host-guest complexes with rotated base pairs, and glyoxal that is known to bind stably to unpaired bases. These differences and additional findings are well-accommodated by the corresponding structures predicted on theoretical grounds.

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“…Depending on the base (pyrimidine or purine) and the sequence context, flipping may occur from either the minor or major groove. The free energy profile for such a reaction cannot be directly measured experimentally, but can be calculated using computational methods [8][9][10][11][12][13][14]. A schematic profile mimicking that obtained from a potential of mean force calculation is shown in Figure 2.…”

Section: The Energetics Of Flipping Bases Computational Studiesmentioning

confidence: 99%

“…It is the purpose of this review to highlight the findings from two DNA repair glycosylases that have benefited most from these approaches. Since several reviews have previously dealt with the subject of enzymatic base flipping, the reader is referred to these for further information [1,2,4,8]. To avoid redundancy, this article emphasizes recent findings that have provided important insights into the nature of the reaction pathway for enzymatic base flipping, and importantly, elucidation of the dynamic properties of DNA that impact the recognition of damaged DNA bases.…”

Section: Introductionmentioning

confidence: 99%

Extrahelical Damaged Base Recognition by DNA Glycosylase Enzymes

Stivers

2008

Chemistry A European J

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The efficient enzymatic detection of damaged bases concealed in the DNA double helix is an essential step during DNA repair in all cells. Emergent structural and mechanistic approaches have provided glimpses into this enigmatic molecular recognition event in several systems. A ubiquitous feature of these essential reactions is the binding of the damaged base in an extrahelical binding mode. The reaction pathway by which this remarkable extrahelical state is achieved is of great interest and even more debate.

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Computational Approaches for Investigating Base Flipping in Oligonucleotides

Cited by 87 publications

References 130 publications

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

The structure of DNA overstretched from the 5′5′ ends differs from the structure of DNA overstretched from the 3′3′ ends

Extrahelical Damaged Base Recognition by DNA Glycosylase Enzymes

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