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  • Recognition and Repair of DNA Damage


    Recognition and Repair of DNA Damage DNA is a reactive molecule that is continually challenged by both endogenous and exogenous insults 1., 2.. Cellular metabolites and their byproducts, environmental toxins, and radiation alter the chemical structure of DNA, producing a wide spectrum of DNA damage. Single and double-strand breaks (DSBs) are generated by hydrolysis of the phosphodeoxyribose backbone, nucleotide mismatches are introduced by replication errors, and nucleobases are modified by alkylation, oxidation, and deamination (Figure 1A). Chemical adducts range in size from a single non-hydrogen 8711 (e.g., 8-oxoguanine, 3-methyladenine) to bulky lesions with helix-distorting properties, such as those produced by polyaromatic hydrocarbons and crosslinking agents. These chemically diverse lesions interfere with normal cellular processes through inhibition of replication, transcription, and chromosome maturation, leading to chromosome rearrangements and instability, cell death, aging, and diseases including cancer [3]. Several DNA repair pathways eliminate specific types of damage from the genome [3]. Pathway choice is dictated in part by the enzymes that recognize or initiate repair of a particular type of damage. By and large, the BER pathway (Figure 1B) eliminates nucleobases with small modifications, abasic sites, and single-strand breaks, whereas nucleotide excision repair (NER; see Glossary) removes bulky, helix-destabilizing lesions. BER is initiated by lesion-specific DNA glycosylases that excise the modified nucleobase from the DNA by catalyzing hydrolysis of the N-glycosidic bond (Figures 1B and 2A). The resulting apurinic/apyrimidinic (AP) site is incised by an AP endonuclease (or a bifunctional DNA glycosylase), generating a 3′-hydroxyl group needed for polymerase-dependent synthesis of new DNA (4., 5., 6. for a detailed overview of BER). Almost every DNA glycosylase, regardless of its specificity or structural architecture, uses a similar overall strategy in which the aberrant nucleotide is flipped out of the duplex and trapped in a nucleobase binding pocket on the protein surface, while the resulting void left in the DNA is filled by one or more intercalating residues that stabilize the extrahelical conformation (Figure 2B,C) 7., 8., 9.. Remodeling of the DNA substrate through bending of the helical axis and widening of the minor groove promotes base flipping by decreasing the energy barrier to basepair opening, while also inducing strain that allows the glycosylase to detect altered base stacking, base pairing, or solvation resulting from chemical modification of the nucleobase 7., 10., 11., 12., 13.. In addition to allowing more stringent substrate recognition, base flipping facilitates excision by enabling catalytic activation of the lesion through chemical complementarity between the active site and the target nucleobase, and by improving reaction geometry between the glycosidic bond and an attacking water molecule (Box 1) [9]. Nucleobase binding pockets are generally too small to accommodate more than a small modification, and thus the discovery of DNA glycosylases capable of removing bulky or crosslinked lesions associated with other types of repair, as well as nontoxic or unmodified bases, has been unexpected. This review describes these new glycosylases, with a focus on the structural mechanisms that enable their activities.
    Self-Resistance to Genotoxic Secondary Metabolites Bacterial secondary metabolites are often used as defense mechanisms in microbial warfare. To withstand the toxicity of their own natural products, antibiotic-producing bacteria require self-resistance mechanisms, such as sequestration, efflux, and degradation of the toxin, and protection and repair of the target (e.g., DNA) [14]. Two target-repair mechanisms for the genotoxins yatakemycin (YTM) and azinomycin B (AZB) were recently found to involve the evolutionarily unrelated DNA glycosylases YtkR2 and AlkZ 15., 16., which are encoded by genes embedded within the ytk and azi biosynthesis clusters 15., 17.. Homologs of these enzymes are present in diverse bacterial species, but only some of these are known to produce antibiotics 16., 18., 19., 20.. Homologs of YtkR2 are also present in archaea and lower eukaryotes. It is unclear whether these organisms encounter compounds similar to YTM and AZB in their environments, or if these homologs have evolved to perform different functions.