Two Glycosylase/Abasic Lyases from Neisseria mucosaThat Initiate DNA Repair at Sites of UV-induced Photoproducts

Abstract

Diverse organisms ranging from Escherichia coli to humans contain a variety of DNA repair proteins that function in the removal of damage caused by shortwave UV light. This study reports the identification, purification, and biochemical characterization of two DNA glycosylases with associated abasic lyase activity from Neisseria mucosa. These enzymes, pyrimidine dimer glycosylase I and II (Nmu-pdg I and Nmu-pdg II), were purified 30,000-and 10,000-fold, respectively. SDS-polyacrylamide gel electrophoresis analysis indicated that Nmu-pdg I is approximately 30 kDa, whereas Nmu-pdg II is approximately 19 kDa. The N-terminal amino acid sequence of Nmu-pdg II exhibits 64 and 66% identity with E. coli and Hemophilus parainfluenzae endonuclease III, respectively. Both Nmu-pdg I and Nmu-pdg II were found to have broad substrate specificities, as evidenced by their ability to incise DNA containing many types of UV and some types of oxidative damage. Consistent with other glycosylase/abasic lyases, the existence of a covalent enzyme-DNA complex could be demonstrated for both Nmu-pdg I and II when reactions were carried out in the presence of sodium borohydride. These data indicate the involvement of an amino group in the catalytic reaction mechanism of both enzymes.The exposure of DNA to UV light results in the formation of several photoproducts including cyclobutane pyrimidine dimers, (6-4) photoproducts, and Dewar photoproducts (1, 2). Cyclobutane pyrimidine dimers are the most common lesions produced by exposure of DNA to short wavelengths of UV light (below 295 nm). This lesion can exist in two main forms as follows: the cis-syn isomer, which is the predominant form, and the trans-syn isomer, which is less common (3). The trans-syn dimer exists as two stereoisomers, trans-syn I and trans-syn II (4). A defect in the repair of pyrimidine dimers can be associated with several biological consequences, including cell death, mutagenesis, and potentially, carcinogenesis. Prokaryotic and eukaryotic organisms possess elaborate mechanisms either for the removal of these lesions (nucleotide excision repair and enzyme-catalyzed photoreversal) or for damage avoidance (recombination), thus enhancing survival and minimizing mutagenesis (reviewed in Refs. 5 and 6).In addition, various organisms and viruses possess a base excision repair (BER) 1 mechanism for the removal of these lesions (Ref. 7 and reviewed in Refs. 8 -10). The initial step in BER is carried out by enzymes known as DNA glycosylases, which recognize and remove the damaged base. Many glycosylases have concomitant abasic (AP) lyase activity and are known as glycosylase/AP lyase enzymes. The prototype of these is endonuclease V (since renamed T4-pdg), which was originally discovered in Escherichia coli that had been infected with the bacteriophage T4.T4-pdg is a well characterized BER enzyme that is specific for cis-syn cyclobutane pyrimidine dimers. In addition, this enzyme has been shown to incise DNA at the sites of trans-syn II dimers (3, 7) and the hydroxyl radical-...

DNA base repair – recognition and initiation of catalysis

“…Sequence homologs of the T4 pyrimidine dimer glycosylase (T4‐Pdg) are also present in distantly related bacterial genomes such as Bordetella , Brucella , Haemophilus , Pasteurella and Prochlorococus . Finally, functional homologs of T4‐Pdg have also been found in Neisseria mucosa , in which Nmu ‐Pdg I and II nick DNA containing several UV‐induced damages as well as some types of oxidative lesions (Nyaga & Lloyd, 2000). These enzymes were formerly termed denV or DNA endonuclease V but the biochemical mechanism is that of DNA glycosylases/AP lyases, and these enzymes should not be confused with the 3′ endonuclease V participating in NIR as described later in this review.…”

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“…ROS can also modulate the activity of proteins and genes that respond to stress, and regulate the genes that are related to cell proliferation, differentiation, and apoptosis. The base excision repair (BER) system recognizes and repairs oxidative dam-aged chromosomal DNA (Nyaga and Lloyd, 2000;Garcia et al, 2000;Mitra et al, 2001). UVB directly produces several kinds of photoproducts in DNA in addition to indirect oxidative DNA damage (Ravanat et al, 2001).…”

Structure of T4 Pyrimidine Dimer Glycosylase in a Reduced Imine Covalent Complex with Abasic Site-containing DNA

“…[6][7][8] Designated endonuclease V in bacteriophage T4 (also T4 pyrimidine dimer glycosylase, T4-Pdg), this enzyme is required for enhanced survival after UV irradiation and functions as a DNA glycosylase with AP lyase activity, primarily at sites in duplex DNA containing cis-syn cyclobutane pyrimidine dimers (CPDs). 9 Pyrimidine dimer-specific nicking activities have also been detected in Micrococcus luteus, [10][11][12] Bacillus sphaericus, 13 Neisseria mucosa 14 and Paramecium bursaria chlorella virus-1. [15][16][17][18] T4-Pdg binds DNA non-specifically in vitro at salt concentrations < 50 mM, and in vivo, it incises all CPDs within plasmid DNA to which it binds prior to dissociation.…”