Mechanism of UV endonuclease V cleavage of abasic sites in DNA determined by carbon-13 labeling

Manoharan, Muthiah; Mazumder, Abhijit; Ransom, Stephen C.; Gerlt, J.A.; Bolton, Philip H.

doi:10.1021/ja00216a074

Cited by 80 publications

(61 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This results in the loss of the base and the formation of a covalent attachment of the enzyme to the 2Ј-deoxyribose sugar moiety of the abasic site, forming an imine (Schiff) base intermediate that can conveniently be trapped in vitro by sodium borohydride (11). The formation of a Schiff base is followed by abstraction of the 2Ј-H and cleavage of the 3Ј-C-O bond through an elimination reaction (12)(13)(14). In yeast and humans, the ⑀-amine group of lysine occupies a place in a helix-hairpin-helix-GPD domain considered critical for non-sequence-specific binding to DNA (see Fig.…”

mentioning

confidence: 99%

Conversion of the Bifunctional 8-Oxoguanine/β-δ Apurinic/Apyrimidinic DNA Repair Activities ofDrosophila Ribosomal Protein S3 into the Human S3 Monofunctional β-Elimination Catalyst through a Single Amino Acid Change

Hegde

Kelley²,

Xu³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Drosophila S3 ribosomal protein has important roles in both protein translation and DNA repair. In regards to the latter activity, it has been shown that S3 contains vigorous N-glycosylase activity for the removal of 8-oxoguanine residues in DNA that leaves baseless sites in their places. Drosophila S3 also possesses an apurinic/apyrimidinic (AP) lyase activity in which the enzyme catalyzes a ␤-elimination reaction that cleaves phosphodiester bonds 3 and adjacent to an AP lesion in DNA. In certain situations, this is followed by a ␦-elimination reaction that ultimately leads to the formation of a single nucleotide gap in DNA bordered by 5-and 3-phosphate groups. The human S3 protein, although 80% identical to its Drosophila homolog and shorter by only two amino acids, has only marginal N-glycosylase activity. Its lyase activity only cleaves AP DNA by a ␤-elimination reaction, thus further distinguishing itself from the Drosophila S3 protein in lacking a ␦-elimination activity. Using a hidden Markov model analysis based on the crystal structures of several DNA repair proteins, the enzymatic differences between Drosophila and human S3 were suggested by the absence of a conserved glutamine residue in human S3 that usually resides at the cleft of the deduced active site pocket of DNA glycosylases. Here we show that the replacement of the Drosophila glutamine by an alanine residue leads to the complete loss of glycosylase activity. Unexpectedly, the ␦-elimination reaction at AP sites was also abrogated by a change in the Drosophila glutamine residue. Thus, a single amino acid change converted the Drosophila activity into one that is similar to that possessed by the human S3 protein. In support of this were experiments executed in vivo that showed that human S3 and the Drosophila site-directed glutamine-changed S3 performed poorly when compared with Drosophila wildtype S3 and its ability to protect a bacterial mutant from the harmful effects of DNA-damaging agents.Aerobic respiration produces electrophilic oxidants known as reactive oxygen species, which are potentially deleterious to the stability and integrity of DNA. Beyond the internal cellular production of reactive oxygen species, the burden of free radical attack on cellular DNA is increased through exposure to ionizing radiation (1), cigarette tar and smoke (2), and the recently discovered presence of stable free radicals associated with particulate matter generated by combustion (3).The impact that free radicals have on DNA is varied and profound. For example, single-and double-strand breaks, along with the production of baseless sites in DNA, are a common result of free radical damage to DNA (4). Oxidatively damaged DNA bases also result from the presence of free radicals; the most notable of such bases is 7, 8-dihydro-8-oxoguanine (8-oxoG). This lesion frequently mispairs with adenine, resulting in G:C 3 T:A transversion mutations (5) that are exceedingly common in somatic mutations found in human cancers and, importantly, are abundant in the tumor supp...

show abstract

mentioning

confidence: 99%

Conversion of the Bifunctional 8-Oxoguanine/β-δ Apurinic/Apyrimidinic DNA Repair Activities ofDrosophila Ribosomal Protein S3 into the Human S3 Monofunctional β-Elimination Catalyst through a Single Amino Acid Change

Hegde

Kelley²,

Xu³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Mechanistic studies have demonstrated that the N-terminal (Thr-2) ␣NH 2 group of the enzyme initiates the glycosylase action by forming an imino intermediate with the C-1Ј of the 5Ј sugar within the pyrimidine dimer (3,14,15). Phosphodiester bond scission occurs by a ␤-elimination mechanism to produce a cleaved site with 5Ј phosphate and 3Ј ␣,␤-unsaturated aldehyde termini (2,6,8). In addition to the catalytic amino group at the N terminus, the acidic residue Glu-23 is essential for both activities (4, 5, 9).…”

mentioning

confidence: 99%

The interaction of T4 endonuclease V E23Q mutant with thymine dimer- and tetrahydrofuran-containing DNA

Latham¹,

Manuel²,

Lloyd³

1995

J Bacteriol

View full text Add to dashboard Cite

The interaction between endonuclease V, the cyclobutane pyrimidine dimer-specific N-glycosylase/abasic lyase from bacteriophage T4, and DNA was investigated by DNase I footprinting methods. The catalytically inactive mutant E23Q was found to interact with a smaller region of DNA at the abasic site analog, tetrahydrofuran, than at a thymine dimer site. Like the wild-type enzyme, the mutant contacted the DNA substrates primarily on the strand opposite the damage. The various complexes examined by footprinting techniques represent distinct points along the catalytic pathway of endonuclease V: before catalysis at a dimer, after N-glycosylase action but before abasic lyase action, and before catalysis at an abasic site. The differences between the footprints of the mutant and wild-type enzymes on both DNA substrates likely represent subtly different conformations within these complexes.Endonuclease V is a 16-kDa N-glycosylase/ abasic (AP) lyase from bacteriophage T4 specific for UV-induced cyclobutane pyrimidine dimers. Mechanistic studies have demonstrated that the N-terminal (Thr-2) ␣NH 2 group of the enzyme initiates the glycosylase action by forming an imino intermediate with the C-1Ј of the 5Ј sugar within the pyrimidine dimer (3,14,15). Phosphodiester bond scission occurs by a ␤-elimination mechanism to produce a cleaved site with 5Ј phosphate and 3Ј ␣,␤-unsaturated aldehyde termini (2,6,8). In addition to the catalytic amino group at the N terminus, the acidic residue Glu-23 is essential for both activities (4, 5, 9). The structure of endonuclease V has been determined to a 1.6-Å (1 Å ϭ 0.1 nm) resolution by X-ray crystallography (12), but little is known about the overall conformation of the protein-DNA complex. In a previous study, DNase I, 1,10-phenanthrolinecopper, and methylation protection assays were used to determine the contacts made between wild-type endonuclease V and a thymine dimer-containing oligonucleotide. To perform these experiments, the imino intermediate was reduced by the addition of NaBH 4 , which results in a dead-end covalent complex (7). The results of the DNase I footprinting of this complex demonstrated that the enzyme interacted with the substrate primarily on the strand opposite the thymine dimer and in a very asymmetric manner. To extend our knowledge about endonuclease V-DNA interactions, in the current study we describe DNase I protection assays using the catalytically inactive endonuclease V mutant, E23Q, bound to both thymine dimer-containing and tetrahydrofuran (THF [an AP site analog])-containing DNA. With the protection patterns displayed by wild-type enzyme covalently trapped to dimer-containing DNA (7), these results allow us to compare the protein-DNA interactions of endonuclease V at different points along its catalytic pathway: before catalysis (E23Q-dimer-containing DNA complex), after the N-glycosylase but before AP lyase action (wild-type enzyme-dimer-containing DNA covalent complex), and at an abasic site (wild-type enzyme-and E23Q-THFcontaining DNA complexes). DNa...

show abstract

“…T4-pdg is known to incise the N-glycosyl bond of the 5Ј-pyrimidine of a dimer and then cleave the sugar-phosphate backbone on the 3Ј side of the abasic site sugar by a ␤-elimination mechanism. Its cleavage product terminates in a 3Ј ␣,␤-unsaturated aldehyde and can be identified by gel electrophoresis (18,19). Treatment of this product with piperidine results in a loss of the modified sugar, leaving a 3Ј-phosphate terminus.…”

Section: Comparison Of Nmu-pdg I Nmu-pdg Ii and T4-pdg Reaction Promentioning

confidence: 99%

“…In the absence of strong reducing agents, the sugar-phosphate backbone is cleaved on the 3Ј side of the abasic site sugar by a ␤-elimination mechanism (lyase reaction). This results in cleavage products with 5Ј-phosphate and 3Ј-␣,␤-unsaturated aldehyde termini, respectively (14,(17)(18)(19). Thus, T4-pdg catalyzes successive N-glycosylase and AP lyase reactions.…”

mentioning

confidence: 99%

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

Nyaga

Lloyd

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

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-...

show abstract

Mechanism of UV endonuclease V cleavage of abasic sites in DNA determined by carbon-13 labeling

Cited by 80 publications

References 0 publications

Conversion of the Bifunctional 8-Oxoguanine/β-δ Apurinic/Apyrimidinic DNA Repair Activities ofDrosophila Ribosomal Protein S3 into the Human S3 Monofunctional β-Elimination Catalyst through a Single Amino Acid Change

Conversion of the Bifunctional 8-Oxoguanine/β-δ Apurinic/Apyrimidinic DNA Repair Activities ofDrosophila Ribosomal Protein S3 into the Human S3 Monofunctional β-Elimination Catalyst through a Single Amino Acid Change

The interaction of T4 endonuclease V E23Q mutant with thymine dimer- and tetrahydrofuran-containing DNA

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

Contact Info

Product

Resources

About