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R P Cunningham

Publications and source records attributed to R P Cunningham.

At least 37 records · Page 2Linked to original sources
PubMed1995

Comparison of the effects of UV irradiation on 5-methyl-substituted and unsubstituted pyrimidines in alternating pyrimidine-purine sequences in DNA.

We previously demonstrated the UV-induced formation of cytosine hydrate in DNA and its deamination product, uracil hydrate, via their release from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated release of thymine hydrate from UV-irradiated poly(dA-dT) was reported. Therefore, we asked whether 5-methylcytosine residues in DNA underwent photohydration and deamination to thymine hydrate in analogy to UV-induced deamination of cytosine. An alternating DNA copolymer containing 5-methylcytosine was irradiated with UVC and incubated with endonuclease III. No 5-methylcytosine hydrate was released. Instead, UV-induced nonenzymatic release of 5-methylcytosine occurred. Similarly, incubation of UV-irradiated poly(dA-dT) with endonuclease III did not release thymine hydrate; nonenzymatic release of thymine occurred. Nonenzymatic release of 5-methylpyrimidines was oxygen dependent, enhanced by ferric ion and inhibited by free radical scavengers. In contrast, photohydration of cytosine was oxygen independent, and only small amounts of cytosine were nonenzymatically released. Thus, 5-methylpyrimidine residues within alternating Pu-Py sequences in DNA do not undergo photohydration, but instead undergo cleavage of their N-glycosyl bonds yielding abasic (AP) sites. The inability to repair such AP sites may explain the UV sensitivity of E. coli xthnfo mutants, which lack AP endonuclease activity. We suggest that N-glycosyl bond cleavage is mediated by radical species formed via transfer of an electron from UV-excited triplet 5-methylpyrimidines to ground state oxygen and/or ferric ions.

DNA↗
PubMed1995

Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.

The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.

Amino Acid Sequence↗
PubMed1995

Structure and function of the multifunctional DNA-repair enzyme exonuclease III.

The repair of DNA requires the removal of abasic sites, which are constantly generated in vivo both spontaneously and by enzymatic removal of uracil, and of bases damaged by active oxygen species, alkylating agents and ionizing radiation. The major apurinic/apyrimidinic (AP) DNA-repair endonuclease in Escherichia coli is the multifunctional enzyme exonuclease III, which also exhibits 3'-repair diesterase, 3'-->5' exonuclease, 3'-phosphomonoesterase and ribonuclease activities. We report here the 1.7 A resolution crystal structure of exonuclease III which reveals a 2-fold symmetric, four-layered alpha beta fold with similarities to both deoxyribonuclease I and RNase H. In the ternary complex determined at 2.6 A resolution, Mn2+ and dCMP bind to exonuclease III at one end of the alpha beta-sandwich, in a region dominated by positive electrostatic potential. Residues conserved among AP endonucleases from bacteria to man cluster within this active site and appear to participate in phosphate-bond cleavage at AP sites through a nucleophilic attack facilitated by a single bound metal ion.

Amino Acid Sequence↗
PubMed1995

Endonuclease III interactions with DNA substrates. 1. Binding and footprinting studies with oligonucleotides containing a reduced apyrimidinic site.

The binding of endonuclease III from Escherichia coli to damaged DNA has been studied using gel shift and footprinting assays. Oligonucleotides containing a reduced apyrimidinic (AP) site were used since reduction of the AP site blocks the beta-elimination reaction catalyzed by the enzyme and yields a noncleavable substrate. The Kobs for a 13-mer carrying a centrally located reduced AP site is (2 x 10(6)-(2 x 10(7) M-1, while the Kobs for a 13-mer with no damage is (4.5 x 10(3)-(3.2 x 10(4) M-1 (approximately a 500-fold difference). Larger oligonucleotides would not enter a gel when endonuclease III was bound so that binding constants to oligonucleotides longer than 13 base pairs could not be determined directly. Competition assays suggest that the Kobs measured for both damaged and undamaged 13-mers is a minimum value and that the Kobs for larger oligonucleotides could be an order of magnitude greater. Fluorescence quenching on related 19-mers yielded a specific binding constant for the 19-mer carrying a centrally located reduced AP site for 4 x 10(7) M-1 and a nonspecific binding constant to an undamaged 19-mer of approximately 10(5) M-1 [Xing, D., Dorr, R., Cunningham, R. P., & Scholes, C. P. (1995) Biochemistry 34, 2537-2544]. Several footprinting reagents were used to determine the size and location of the endonuclease III binding site on damaged oligonucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Endonuclease III interactions with DNA substrates. 2. The DNA repair enzyme endonuclease III binds differently to intact DNA and to apyrimidinic/apurinic DNA substrates as shown by tryptophan fluorescence quenching.

We have measured the fluorescence of the DNA repair enzyme endonuclease III to discover perturbation to its tryptophans by undamaged DNA and AP (apyrimidinic or apurinic) DNA and to estimate binding affinity for intact and AP DNAs. Endonuclease III has two tryptophans, Trp132 in a helix-hairpin-helix region of possible flexibility near the active site for AP lyase activity and Trp178 in the domain containing the iron-sulfur center of endonuclease III; Trp132 is the more solvent-accessible tryptophan [Kuo, C.-F., McRee, D. E., Fisher, C. L., O'Handley, S. F., & Cunningham, R. P. (1992) Science 258, 434-440]. The fluorescence emission peak wavelength near 350 nm (excitation at 290 nm) indicated an exposure of the fluorescing tryptophans to a polar environment. Quenching of tryptophan fluorescence by iodide demonstrated that there are indeed two tryptophans which are differently accessible to anionic quencher. Significant (approximately 60%) fluorescence quenching occurred when endonuclease III was titrated with high molecular weight duplex undamaged poly(dAdT). The apparent second-order nonspecific binding constant to poly(dAdT) was 4 x 10(7) M-1, and there were approximately 12 base pairs per endonuclease III binding site for binding to poly(dAdT). This nonspecific binding to duplex DNA had ionic character, and there was no fluorescence quenching brought on by single-stranded DNA. A comparison between fluorescence quenching titrations of high molecular weight duplex DNA and undamaged duplex 19-mer oligonucleotide showed that the binding constant to the high molecular weight DNA was approximately 400-fold larger than to the undamaged 19-mer.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

DNA repair proteins.

DNA repair proteins act to correct mutagenic and toxic DNA damage, which can lead to cancer, aging and death. These proteins and their mechanisms of action have been found to be widely conserved between species, often from bacteria to man. Structural and biochemical studies on several bacterial enzymes involved in direct reversal and base excision repair have provided insights into the molecular basis of the recognition of damaged DNA and have also highlighted the novel roles that transition metals play in DNA repair.

Amino Acid Sequence↗
PubMed1995

Identification of critical active-site residues in the multifunctional human DNA repair enzyme HAP1.

All organisms express dedicated repair enzymes for counteracting the cytotoxic and mutagenic potential of apurinic/apyrimidinic (AP) lesions, which would otherwise pose a serious threat to genome integrity. We present the predicted three-dimensional structure of the major human AP site-specific DNA repair endonuclease, HAP1, and show that an aspartate/histidine pair, in conjunction with a metal ion-coordinating glutamate residue, are critical for catalyzing the multiple repair activities of HAP1. We suggest that this catalytic mechanism is conserved in certain reverse transcriptases, but is distinct from the two metal ion-mediated mechanism defined for other hydrolytic nucleases.

Amino Acid Sequence↗
PubMed1994

Effect of pH and temperature on the stability of UV-induced repairable pyrimidine hydrates in DNA.

UV irradiation of cytosine yields 6-hydroxy-5,6-dihydrocytosine (cytosine hydrate) whether the cytosine is in solution as base, nucleoside, or nucleotide or on the DNA backbone. Cytosine hydrate decomposes by elimination of water, yielding cytosine, or by irreversible deamination, yielding uracil hydrate, which, in turn, decomposes by dehydration yielding uracil. To determine how pH and temperature affect these decomposition reactions, alternating poly(dG-[3H]dC) copolymer was irradiated at 254 nm and incubated under different conditions of pH and temperature. The cytosine hydrate and uracil hydrate content of the DNA was determined by the use of Escherichia coli endonuclease III, which releases pyrimidine hydrates from DNA by virtue of its DNA glycosylase activity. Uracil content was determined by using uracil-DNA glycosylase. The rate of decomposition of cytosine hydrate to cytosine was determined at 4 temperatures at pH 3.1, 5.4, and 7.4. The Ea was determined from the rates by using the Arrhenius equation and proved to be the same at pH 5.4 and 7.4, although the decomposition rate at pH 5.4 was faster at all temperatures. At pH 3.1, the Ea was reduced. These results suggest that the dehydration reaction is affected by two discrete protonations, most probably of the N-3 and the OH group of C-6 of cytosine hydrate. The deamination of cytosine hydrate to uracil hydrate was maximal at pH 3.1 at all temperatures. The doubly protonated cytosine hydrate probably is the common intermediate for both competing decomposition reactions, explaining why cytosine hydrate is prone to deamination at acid pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosine↗
PubMed1994

Structure and function of the DNA repair enzyme exonuclease III from E. coli.

The three-dimensional structure of exonuclease III, the major AP DNA repair endonuclease of Escherichia coli, has been determined using x-ray crystallographic methods at 2.7 A resolution. The atomic model was fit to an electron density map calculated with phases obtained from three isomorphous heavy atom derivatives. The overall chain fold of exonuclease III is that of a compact alpha,beta-protein of dimensions 55 by 50 by 45 A. The pair of extended beta-pleated sheets pack against each other in an approximately parallel fashion to form the hydrophobic core of a four-layered sandwich structure. These beta sheets are flanked by four alpha-helices that form the outer two layers of the fold. The individual strands of the beta-sheets are in a mostly antiparallel configuration and are linked by extensive loop regions that connect adjoining strands. The structure contains internal symmetry with the two extended beta-sheets and four alpha-helices related by a pseudo-twofold axis running approximately down the center of the two sheets. This internal symmetry is not mirrored in the structure of the loop regions, nor is it detectable within the amino acid sequence. There is a "groove" between the beta-sheets at one end of the molecule that is bordered by several of the exposed loop regions and may be significant for DNA binding.

Amino Acid Sequence↗
PubMed1994

New substrates for old enzymes. 5-Hydroxy-2'-deoxycytidine and 5-hydroxy-2'-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2'-deoxyuridine is a substrate for uracil DNA N-glycosylase.

5-Hydroxy-2'-deoxycytidine (5-OHdC) and 5-hydroxy-2'-deoxyuridine (5-OHdU) are major products of oxidative DNA damage with mutagenic potential. Until now, no enzymatic activity responsible for their removal has been identified. We report here that both 5-OHdC and 5-OHdU are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase (FPG). 5-OHdU is also a substrate for uracil DNA N-glycosylase. Consistent with their mechanisms of action on previously described substrates, endonuclease III removes 5-OHdC and 5-OHdU via a N-glycosylase/beta-elimination reaction, FPG follows a N-glycosylase/beta,delta-elimination reaction, and uracil N-glycosylase removes 5-OHdU by N-glycosylase action leaving behind an abasic site. Endonuclease III removes both lesions more efficiently than FPG, and both endonuclease III and FPG remove 5-OHdC slightly more efficiently than 5-OHdU. Uracil DNA N-glycosylase removes 5-OHdU more efficiently than the other two enzymes and has no activity on 5-OHdC even when present in great excess. Analysis of crude extracts obtained from wild type and endonuclease III deletion mutants of E. coli correlated well with data obtained with the purified enzymes.

Base Sequence↗
PubMed1993

Purification, crystallization and space group determination of DNA repair enzyme exonuclease III from E. coli.

Escherichia coli exonuclease III possesses multiple catalytic activities: (1) a nucleotidyl hydrolase activity cutting 5' to apurinic/apyrimidinic sites and urea residues in DNA; (2) a 3' to 5' exonuclease activity specific for double-stranded DNA; (3) a RNase H activity preferentially degrading the RNA strand of a DNA.RNA hybrid and (4) an activity that can remove a number of 3' termini from duplex DNA including 3' phosphates, 3' phosphoglycolate residues, 3' phosphoglycolaldehyde residues and 3' trans-4-hydroxy-2-pentenal-5-phosphate residues. These multiple activities make exonuclease III a major enzyme in the base excision repair pathway for DNA damage. We have purified exonuclease III and grown crystals by the vapor diffusion method using polyethylene glycol 4000 as the precipitant. Buffers were found to have profound effects on crystallization with high concentrations of imidazole/malate buffer (0.4 M to 1.0 M) yielding larger crystals with less twinning. The crystals belong to the space group P3(1)21 or its enantiomorph P3(2)21 with unit cell dimensions of a = b = 107.8 A, c = 42.2 A, alpha = beta = 90 degrees, gamma = 120 degrees, have one 31 kDa monomer per asymmetric unit and diffract to 1.6 A. These crystals are stable to X-rays and suitable for high resolution structure determination.

Crystallization↗
PubMed1993

Molecular recognition in DNA-binding proteins and enzymes.

The molecular basis for the specificity and activity of protein-DNA interactions is currently being established from the combination of results on the structure and biochemistry of DNA-binding proteins and enzymes. Data detailed in the 12 most recent studies on DNA-binding protein and enzyme structures, including the major advances in the elucidation of enzyme-mediated DNA-repair processes, have both increased understanding of DNA recognition and enhanced prospects for the design of novel DNA-binding proteins in the future.

Biotechnology↗
PubMed1992

Atomic structure of the DNA repair [4Fe-4S] enzyme endonuclease III.

The crystal structure of the DNA repair enzyme endonuclease III, which recognizes and cleaves DNA at damaged bases, has been solved to 2.0 angstrom resolution with an R factor of 0.185. This iron-sulfur [4Fe-4S] enzyme is elongated and bilobal with a deep cleft separating two similarly sized domains: a novel, sequence-continuous, six-helix domain (residues 22 to 132) and a Greek-key, four-helix domain formed by the amino-terminal and three carboxyl-terminal helices (residues 1 to 21 and 133 to 211) together with the [4Fe-4S] cluster. The cluster is bound entirely within the carboxyl-terminal loop with a ligation pattern (Cys-X6-Cys-X2-Cys-X5-Cys) distinct from all other known [4Fe-4S] proteins. Sequence conservation and the positive electrostatic potential of conserved regions identify a surface suitable for binding duplex B-DNA across the long axis of the enzyme, matching a 46 angstrom length of protected DNA. The primary role of the [4Fe-4S] cluster appears to involve positioning conserved basic residues for interaction with the DNA phosphate backbone. The crystallographically identified inhibitor binding region, which recognizes the damaged base thymine glycol, is a seven-residue beta-hairpin (residues 113 to 119). Location and side chain orientation at the base of the inhibitor binding site implicate Glu112 in the N-glycosylase mechanism and Lys120 in the beta-elimination mechanism. Overall, the structure reveals an unusual fold and a new biological function for [4Fe-4S] clusters and provides a structural basis for studying recognition of damaged DNA and the N-glycosylase and apurinic/apyrimidinic-lyase mechanisms.

Bacterial Proteins↗
PubMed1992

Crystallization and crystallographic characterization of the iron-sulfur-containing DNA-repair enzyme endonuclease III from Escherichia coli.

Endonuclease III from Escherichia coli is an iron-sulfur enzyme possessing both DNA N-glycosylase and apurinic/apyrimidinic lyase activities. It could serve to repair damaged thymine residues in DNA via base excision-repair. We have crystallized endonuclease III by a combination of dialysis and seeding techniques after exploration of a wide variety of precipitants which failed to yield macroscopic crystals. Important features of the optimized crystallization include: the use of 5 to 10% glycerol, a temperature of 15 degrees C, controlled dialysis to decrease ionic strength and macroseeding using a 200 mM-NaCl transfer buffer to dissolve microcrystalline contamination. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 48.5 A, b = 65.8 A, c = 86.8 A, alpha = beta = gamma = 90 degrees, have one 23 kDa monomer per asymmetric unit, and diffract to 1.84 A. A native anomalous Patterson map located the iron-sulfur cluster and reaffirmed its existence. The reported crystallization procedures ensure an ample supply of crystals for the extensive heavy-atom derivative search necessary for this labile iron-sulfur enzyme. The elucidation of endonuclease III structure will facilitate not only the understanding of glycosylase and lyase mechanisms but also the structure and function of this new class of iron-sulfur proteins.

Bacterial Proteins↗
PubMed1992

The role of the iron-sulfur cluster in Escherichia coli endonuclease III. A resonance Raman study.

Resonance Raman spectroscopy has been used to investigate the function and properties of the iron-sulfur cluster in Escherichia coli endonuclease III. Resonance Raman spectra in the Fe-S stretching region are indicative of a [4Fe-4S]2+ cluster with complete cysteinyl sulfur coordination, and vibrational assignments are made by analogy with bacterial ferredoxins. Minor changes in the vibrational frequencies of the modes primarily involving Fe-S(Cys) stretching accompany the binding of the inhibitor thymine glycol or an oligonucleotide containing a reduced apyrimidinic site. These changes are consistent with perturbation of the orientation of the ligating cysteinyl residues and rule out the possibility that the [4Fe-4S] cluster is directly involved with substrate or inhibitor binding. It is concluded that a structural role is most likely for the [4Fe-4S] cluster in endonuclease III.

Base Sequence↗