We first discuss the fundamental physical problems associated with locating rare damaged bases in DNA so that the mechanistic solutions taken by these enzymes can be placed in a contextual framework. Here we highlight recent studies that are beginning to reveal the basic mechanistic elements in damaged base detection by several DNA glycosylases. These intriguing aspects of damage detection, and the mechanistic insights provided by these new approaches, are the primary focus of this review. This realization has been the driving force for the development of new structural and biophysical methods capable of elucidating transient intermediates that occur very early in the pathway, and for understanding the important mechanism of diffusional encounter with a damage site. Notwithstanding with these successes, a mechanistic understanding of the extended reaction coordinate for base flipping has not been forthcoming by documenting interactions present in structural snapshots of the reaction endpoint. Such structural efforts have been extraordinarily successful, and coordinates for over 21 enzyme-DNA complexes have been deposited in the protein data bank. Initial structural investigations into enzymatic base flipping focused on elucidating the final extrahelical state, where the damaged base was rotated fully into the enzyme active site and poised for glycosidic bond cleavage. These costs include breaking of Watson-Crick hydrogen bonds, the disruption of aromatic stacking interactions with adjacent bases, and large perturbations in the phosphate torsion angles around the flipped base. The overall reaction is driven forward solely by the use of enzyme binding energy for DNA, which is used to pay for the significant energetic costs of extracting a base from the DNA base stack. Summary of substrate specificities, structural characterizations with bound DNA, and kinetic studies on representative DNA repair glycosylases Base flipping involves one of the most extended and improbable reaction trajectories in biology. This process has been called either base or nucleotide “flipping” by various investigators (, ), and connects the damage encounter event with the catalytic step of bond scission. Although these enzymes fall into different structural classes, and each is specialized for the detection and removal of different types of damaged bases, with the sole exception of pyrimidine dimer DNA glycosylase, these enzymes have converged on a single mechanistic solution for damaged base recognition and excision: rotation of the damaged base from the DNA base stack into a sequestered active site pocket where chemistry occurs. The cellular sentinels at this first step are the remarkable DNA glycosylase enzymes. The initiating step in this pathway begins with the enzymatic hydrolysis of the glycosidic bond that attaches the damaged base to the deoxyribose phosphate DNA backbone, setting the stage for the multistep base excision repair process to begin. The problem of enzymatic detection of a single damaged base in the context of a vast genome of nearly isomorphous undamaged bases has intrigued the DNA repair community virtually since the discovery of the DNA base excision repair pathway. 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. 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). 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. 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 refer to as the search complex (SC). Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Gldirect 5 0 2 Fully Executed 3,1/5 9983 votesĪ fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA.
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