Detection of damaged DNA bases by DNA glycosylase enzymes

Abstract

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Figure 1

The DNA base excision repair pathway begins with the encounter of a rare genomic damaged base (X*) by a damage specific DNA glycosylase. These enzymes hydrolytically excise the damaged base and begin the extended process of replacing it with the original undamaged base (X).

Figure 2

The extended reaction coordinate for extrahelical recognition of undamaged and damaged bases by uracil DNA glycosylase (UNG), and the bacterial and human 8-oxoguanine DNA glycosylases (MutM and hOGG1). Each of these enzymes excises its cognate damaged base through a multistep base flipping reaction coordinate beginning with one or more early interrogation complexes (IC) where normal and damaged bases are inspected, and ending with a fully extrahelical excision complex (EC) poised for glycosidic bond cleavage of the damaged base. The trajectory for base flipping is shown viewing down the helical axis (top) and from an orthogonal perspective in which the enzyme has been removed for clarity (bottom).

Figure 3

Damaged base recognition involves three discrete enzyme DNA complexes. The search complex (SC) is a loose nonspecific complex that is competent for hopping and sliding along the DNA chain. The interrogation complex (IC) is competent for extrahelical or intrahelical interrogation of damaged and undamaged basepairs. The excision complex (EC) is specific for binding of an extrahelical damaged base.

Figure 4

Damaged base recognition involves both three dimensional diffusion and one dimensional DNA sliding (see text and eqs 2 and 3 for definitions).

Figure 5

The free energy surface for enzymatic base flipping involves a DNA bending and base rotation reaction coordinate. Different enzymes show different degrees of progression along each axis during the process of flipping (Fig. 2). The progression of hOGG1 along both coordinates is qualitatively depicted in the free energy surface shown. The trajectory followed by UNG involves a greater progression along the rotation coordinate as compared to bending, while that of MutM may involve full bending before any rotation occurs (Fig. 2).

Figure 6

Normal mode analysis and NMR dynamic data suggest an open (left) to closed (right) transition in UNG during the interrogation of DNA for damage (see text). The open state is envisioned as competent for DNA sliding and the closed conformation (1OXM, Fig. 2) is competent for base interrogation.

Figure 7

Thermally induced flipping of a thymidine base results in an open state that closely resembles the conformation of thymine when bound in the exosite of UNG (see text). The figure is a view down the DNA helical axis, and depicts the conformation of intrahelical thymine in B DNA (blue), the minimum extrahelical state of thymine that is required for imino proton exchange (yellow), and the conformation of thymine when bound to the exosite of UNG (red). To facilitate visual comparisons, the bases have been projected onto a common plane, and the syn-conformation of thymine in the exosite structure is shown in the anti form for ease of comparison with the other two states. For reference, the circle depicts the circumference described by the DNA sugar phosphate backbone.