UV endonuclease V from bacteriophage T4 catalyzes DNA strand cleavage at aldehydic abasic sites by a syn .beta.-elimination reaction

237

335

Oxidative DNA damage is generated by a variety of environmental and endogenous agents, including ionizing radiation, certain chemicals, and products of aerobic metabolism (1). 8-oxoG 1 is one of the most abundant forms of oxidative DNA damage (2). Due to its ability to form a Hoogstein-type base pair with adenine (3), 8-oxoG is miscoding (4) and mutagenic, resulting in G3 T transversions in bacterial and eukaryotic cells (5, 6). The potential harmful effects of this lesion are avoided by base excision repair. In Escherichia coli, formamidopyrimidine-DNA glycosylase (Fpg, EC 3.2.2.23) removes 8-oxoG, Me-FaPy, and several structurally related lesions from damaged DNA (7,8). Fpg is a component of the "GO system" that includes MutY, a mismatch adenine-DNA glycosylase, and MutT, an 8-oxodGTPase (9, 10); E. coli strains deficient in any of these genes are strong mutators (11).Fpg shares significant sequence homology with endonuclease VIII (Nei) of E. coli (12). Both proteins belong to a family unrelated by sequence or tertiary structure to a larger family of DNA glycosylases, for which the prototype is endonuclease III (Nth) (13,14). The substrate specificity of Fpg differs significantly from Nei (7, 8, 15) but closely resembles that of the eukaryotic 8-oxoguanine-DNA glycosylase, Ogg1, a member of the Nth family (14,16,17). Fpg also possesses AP lyase activity, nicking the phosphodiester backbone of DNA at the site of the lesion. Base excision by Fpg is followed immediately by two ␤-elimination steps, resulting in a single nucleotide gap flanked by phosphate termini (7). A Schiff base intermediate, involving Pro-1 of the enzyme and C1Ј of the damaged nucleotide, forms early in the reaction sequence and can be reductively trapped by treatment with NaBH 4 forming a stable covalent complex (18,19). The mechanism of cleavage is similar to that of Nei (15,20), but not to that of Ogg1 where only one ␤-elimination occurs, and the efficiency of the elimination step is very low compared with base excision (16,17).Comparing the structures of Fpg, Nei, and Ogg1 provides a unique opportunity to analyze features of damage recognition and catalysis common to DNA glycosylases/AP lyases. The presence of DNA enhances the analytic power of the model by revealing the precise nature of enzyme-DNA interactions. The structure of the human Ogg1 catalytic domain complexed to DNA has been solved (21, 22), as has the structure of E. coli Nei covalently cross-linked to DNA by NaBH 4 (23). The structure of Fpg from Thermus thermophilus HB8 (Tth-Fpg) has recently been solved in the absence of DNA (24). Although mechanisms for lesion recognition and catalysis by Fpg have been suggested on the basis of this structure and on earlier biochemical studies of E. coli Fpg (8,18,24,25), many questions remain unanswered regarding the mode of Fpg-DNA interactions and the catalytic reaction mechanism of this important DNA repair protein.To investigate the mechanisms of Fpg-DNA interactions, we have utilized NaBH 4 reduction of the Schiff base intermediate t...

Section: Resultscontrasting

confidence: 60%

Structure of Formamidopyrimidine-DNA Glycosylase Covalently Complexed to DNA

Gilboa¹,

Zharkov²,

Golan³

et al. 2002

237

335

“…Assay for Cleavage of AP Site-containing Double-stranded DNA by Human DNA Polymerase ␤ and AP EndonucleaseBoth ␤-pol and AP endonuclease cleave double-stranded DNA containing an AP site (4,(13)(14)(15)(16)32). Whereas the ␤-pol-catalyzed reaction is via ␤-elimination incising the AP site on the 3Ј-side of the sugar, AP endonuclease hydrolyzes the phosphodiester bond 5Ј to the AP site sugar ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Human DNA Polymerase β Deoxyribose Phosphate Lyase

Prasad¹,

Beard²,

Strauss³

et al. 1998

196

166

DNA polymerase ␤ (␤-pol) cleaves the sugar-phosphate bond 3 to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5-incised by AP endonuclease, resulting in a 5 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the aminoterminal 8-kDa domain of ␤-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of ␤-pol are similar. However, k cat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The fulllength enzyme and the 8-kDa domain efficiently remove the 5 dRP from a preincised AP site in the absence of Mg 2؉ , and the pH profiles of ␤-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys 72 ) as the Schiff base nucleophile during lyase chemistry.The cellular genome suffers extensive damage from exposure to ultraviolet light and ionizing radiation and also from alkylating agents and reactive oxygen species that accumulate in cells due to environmental stress and natural metabolic processes (1). These DNA lesions occur at a frequency far too high to be compatible with life unless they are repaired to allow faithful function and reproduction of the genome. To remove such damage and restore the integrity of the genome, specific DNA repair pathways have evolved. The major pathway, which protects cells from the deleterious effects of nucleotide base DNA damage, is known as DNA base excision repair (BER). BER is initiated by removal of a damaged or inappropriate base residue in DNA by cleavage of the N-glycosidic bond (1, 2). If not repaired, the resulting abasic or apurinic/apyrimidinic (AP) site may result in mutations, altered gene expression, chromosome breakage, apoptosis, and/or cell death.Both procaryotes and eucaryotes possess class II AP endonucleases that cleave 5Ј to an AP site leaving a 3Ј-hydroxyl and a 5Ј 2-deoxyribose 5-phosphate (dRP) in the nicked DNA (Fig. 1). In Escherichia coli, two major AP endonuclease genes, xth and nfo, have been identified encoding exonuclease III and endonuclease IV, respectively (3, 4). Homologous genes to xth have been cloned from many sources, including Drosophila melanogaster and human cells (5-7). The major AP endonuclease in yeast, APN1, was shown to be an endonuclease IV homologue (8). These class II AP endonucleases also possess 3Ј-phosphodiesterase and 3Ј-phosphatase activities, and some members of the xth family carry 3Ј to 5Ј exonuclease activity (9). Subsequent to AP endonuclease cleavage in BER, the cleaved AP site must be processed further (Fig. 1). The enzymatic removal of the sugar-phosphate residue ...

“…When these glycosylases also catalyze the breakage of the sugar phosphate backbone by ␤-elimination, they leave a 3Ј-␣, ␤-unsaturated aldehyde, and a 5Ј-phosphate (36,37). In some DNA glycosylases with associated AP lyase activity, there is also a ␦-elimination that is observed at the AP site leaving a 3Ј-phosphate end (38,39).…”

Section: Discussionmentioning

confidence: 99%

Identification of the Structural and Functional Domains of MutY, an DNA Mismatch Repair Enzyme

Manuel

Czerwinski

Lloyd

1996

The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26-and 13-kDa fragments. NH 2 -terminal sequencing showed that the p13 domain begins at Gln 226 , indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A⅐G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met 1 -Trp 216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.Proteins are constructed on a modular basis, and frequently these modules or domains are known to have unique functions. Isolation and characterization of these domains provide significant insight into the relationship between particular structural elements of the enzyme and its various activities. The mutY gene of Escherichia coli encodes a 39.1-kDa DNA mismatch repair protein. A significant portion of this protein is homologous to the 26.3-kDa E. coli endonuclease III. These two proteins are 66.3% similar and 23.8% identical over a 181-amino acid region (1). Another enzyme with sequence similarity to MutY is the product of the pdg gene in Micrococcus luteus, which recognizes and incises DNA containing cyclobutane pyrimidine dimers (2). The three enzymes mentioned above contain a [4Fe-4S] 2ϩ cluster, coordinated by four cysteine residues which are perfectly conserved in all three proteins.In contrast to their structural similarities, the functional properties of the above three DNA repair proteins are very different. MutY recognizes and removes the undamaged adenine mispaired with guanine, cytosine, 8-oxo-7,8-dihydro-2Ј-deoxyguanine (8-oxo-dG) 1 and 8-oxo-7,8-dihydro-2Ј-deoxyadenine (3) and is also reported to have AP endonuclease activity (4, 5). In vitro experiments show that MutY also recognizes and removes adenine analogs when they are paired with guanine (5).2 Endonuclease III primarily removes oxidized pyrimidines from DNA (6), and the pdg gene product in M. luteus repairs UV-induced pyrimidine dimer lesions in DNA (2). Thus, despite their structural similarities, these DNA repair enzymes have different substrate specificity. The catalytic mechanism of endonuclease III in...