Extrahelical damaged base recognition by DNA glycosylase enzymes - PubMed (original) (raw)

Review

Extrahelical damaged base recognition by DNA glycosylase enzymes

James T Stivers. Chemistry. 2008.

Abstract

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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Figures

Figure 1

DNA deformations occuring during the process of enzymatic base flipping. The unfavorable energetic events during the reaction are listed. The enzyme must pay for these costs through the use of favorable binding energy, which would be expected increase over the reaction pathway. The extrahelical conformation on the right was extracted from the complex of uracil DNA glycosylase bound to uracilated DNA (pdb 1EMH).

Figure 2

Reaction coordinate (schematic) for spontaneous base pair opening in B DNA through a 180° rotation along a major groove pathway as determined in potential of mean force calculations [–11]. Characteristic opening equilibria (_k_op/_k_cl), and opening and closing rates (_k_op and _k_cl) for G:C and A:T base pairs are noted (T = 15 °C) [15,17]. The bracket denotes a population of isoenergetic out conformations that are in rapid fluctation.

Figure 3

UNG binds more tightly to T/X (or U/X) base pairs that have large opening equilibrium constants: X = D (diaminopurine), A. (adenine), and N (nebularine). (a) In this series of base pairs, the number of hydrogen bonds in the T/X pair is incrementally decreased from three to one while keeping the shape and electronic properties of the X partner constant. (b) The opening equilibrium constant was measured using NMR imino proton exchange,[19] and then compared with the dissociation constant for UNG binding to each construct (Krosky and Stivers, unpublished).

Figure 4

Strategies for trapping unstable intermediates during base flipping (see text). (a) Disulfide crosslinking. (b) Reaction coordinate tuning.

Figure 5

Intermediates on the base flipping pathways of hOGG1 and UNG[28, 23]. (a) The exo-site complex of hOGG1 with an extrahelical guanine (blue) obtained by disulfide crosslinking technology (left). The fully extrahelical complex with 8-oxoG is shown on the right for comparison [30]. (b) The early exo-site complex of hUNG with an extrahelical thymine (blue) obtained using the reaction coordinate tuning method. The fully extrahelical complex with uracil is shown on the right [33].

Figure 6

The reaction coordinate for uracil flipping by UNG. The microscopic rate constants have been calculated by combining NMR[19,34] and rapid kinetic measurements[24,25]. The profile pertains to 25 °C. The structures are: free human UNG (pdb 1AKZ), intermediate 1 (encounter complex with B DNA, model)[23], intermediate 2 (partially flipped intermediate state, pdb 2OXM), intermediate 3 (detected kinetically, no structural model)[20,25], final flipped state (pdb 1EMH)[33]. Since the rates by neccesity were obtained using different substrates and by extrapolation of the base pair opening rates to 25 °C, the values should only be considered best approximations.

Figure 7

Possible mechanisms for enzymatic recognition of an extrahelical base with a short extrahelical lifetime. A pathway involving bimolecular collision of the enzyme with the DNA base while it exists in an extrahelical conformation is not kinetically competent[34]. Rapid intramolecular transfer of the enzyme along the DNA bypasses the kinetic problem of diffusion and allows the enzyme to rapidly scan short lengths of the DNA duplex before dissociation.

Scheme 1

Reactants and products of several enzymes that use a base flipping mechanism. R2 = (CH2)2CHNH2CO2H, Ad = 5′-adenosyl

References

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Extrahelical damaged base recognition by DNA glycosylase enzymes - PubMed (original) (raw)