Partial purification and characterization of an endonuclease from spinach that cleaves ultraviolet light-damaged duplex DNA

An endonuclease that cleaves ultraviolet light (UV)-damaged, supercoiled plasmid DNA was partially purified from spinach leaves (Spinacia oleracea) by a series of column chromatography steps. Dialysis of the enzyme against EDTA resulted in a greater than 90% loss of activity which could be fully restored following the addition of Zn2+, suggesting that divalent cations are associated with the active enzyme. The spinach endonuclease cleaved duplex, UV-damaged, end-labelled DNA of defined sequence at positions of adenine in the presence of salt (KH2PO4 or NaCl) concentrations of 50 mM or higher. Cleavage of UV-irradiated DNA was dose-dependent and increased steadily within a fluence range of 50-10,000 J/m2. The UV damage requirement and adenine cleavage specificity could be eliminated with lower salt concentrations (0-25 mM), suggesting that the endonuclease recognizes and incises single-stranded DNA. The properties of this enzyme, which we have termed nuclease SP, suggest that it may mediate a role in DNA repair and/or recombination processes in spinach.     

Purification and properties of nuclease SP.

Single-strand-specific nucleases are a diverse and important group of enzymes that are able to cleave a variety of DNA structures present in duplex molecules. Nuclease SP, an enzyme from spinach, has been purified to apparent homogeneity, allowing for the unambiguous characterization of a number of its physical properties as well as its DNA strand cleavage specificities. The effects of ionic strength, pH, divalent metal cations, and temperature on nuclease SP activity have been examined in detail. Nuclease SP was found to be quite thermostable and could be stimulated by Co2+. In addition, the cleavage of UV-damaged and undamaged supercoiled plasmid substrates under a variety of conditions suggests that at least two types of structures are recognized and processed by nuclease SP: UV photoproduct-induced distortions and unwound "nuclease hypersensitive sites". These studies indicate that nuclease SP is functionally related to other single-strand-specific nucleases and is a potential enzymatic tool for probing and manipulating various types of DNA structures.     

T4 DNA polymerase (3''-5'') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example.

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.     

Sequence effect on incision by (A)BC excinuclease of 4NQO adducts and UV photoproducts.

Nucleotide excision repair in Escherichia coli is initiated by (A)BC excinuclease, an enzyme which incises DNA on both sides of bulky adducts and removes the damaged nucleotide as a 12-13 base long oligomer. The incision pattern of the enzyme was examined using DNA modified by 4-nitroquinoline 1-oxide (4NQO) and UV light. Similar to the cleavage pattern of UV photoproducts and other bulky adducts, the enzyme incises the 8th phosphodiester bond 5' and 5th phosphodiester bond 3' to the 4NQO-modifed base, primarily guanine. The extent of DNA damage by these agents was determined using techniques which quantitatively cleave the DNA or stop at the site of the adduct. By comparison of the intensity of gel bands created by (A)BC excinuclease and the specific cleavage at the damaged site, the efficiency of (A)BC excinuclease incision at 13 different 4NQO-induced adducts and 13 different photoproducts was determined by densitometric scanning. In general, incisions made at 4NQO-induced adducts are proportional to the extent of damage, though the efficiency of cutting throughout the sequence tested varies from 25 to 75%. Incisions made at pyrimidine dimers are less efficient than at 4NQO-adducts, ranging from 13 to 65% incision relative to modification, though most are around 50%. The two (6-4) photoproducts within the region tested are incised more efficiently than any pyrimidine dimer.     

Reduced sulfhydryls maintain specific incision of BPDE-DNA adducts by recombinant thermoresistant Bacillus caldotenax UvrABC endonuclease

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. Escherichia coli UvrABC endonuclease can incise DNA containing UV photoproducts and bulky chemical adducts. The limited stability of the E. coli UvrABC subunits leads to difficulty in estimating incision efficiency and quantitative adduct detection. To develop a more stable enzyme with greater utility for the detection of DNA adducts, thermoresistant UvrABC endonuclease was cloned from the eubacterium Bacillus caldotenax (Bca) and individual recombinant protein subunits were overexpressed in and purified from E. coli. Here, we show that Bca UvrC that had lost activity or specificity could be restored by dialysis against buffer containing 500 mM KCl and 20mM dithiothreitol. Our data indicate that UvrC solubility depended on high salt concentrations and UvrC nuclease activity and the specificity of incisions depended on the presence of reduced sulfhydryls. Optimal conditions for BCA UvrABC-specific cleavage of plasmid DNAs treated with [3H](+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (1-5 lesions/plasmid) were developed. Preincubation of substrates with UvrA and UvrB enhanced incision efficiency on damaged substrates and decreased non-specific nuclease activity on undamaged substrates. Under optimal conditions for damaged plasmid incision, approximately 70% of adducts were incised in 1 nM plasmid DNA (2 BPDE adducts/5.4 kbp plasmid) with UvrA at 2.5 nM, UvrB at 62.5 nM, and UvrC at 25 nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.     

Restriction enzyme cleavage of ultraviolet-damaged simian virus 40 and pBR322 DNA

Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases.     

Substrate specificity of ultraviolet DNA endonuclease (UVDE/Uve1p) from Schizosaccharomyces pombe.

Schizosaccharomyces pombe ultraviolet DNA endonuclease (UVDE or Uve1p) has been shown to cleave 5' to UV light-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). This endonuclease is believed to function in the initial step in an alternative excision repair pathway for the removal of DNA damage caused by exposure to UV light. An active truncated form of this protein, Delta228-Uve1p, has been successfully overexpressed, affinity purified and partially characterized. In the present study we present data from a detailed substrate specificity trial. We have determined that the substrate range of Uve1p is much greater than was originally believed. We demonstrate that this DNA damage repair protein is capable of recognizing an array of UV-induced DNA photoproducts (cis-syn-, trans-syn I- and trans-syn II CPDs, 6-4PP and Dewar isomers) that cause varying degrees of distortion in a duplex DNA molecule. We also demonstrate that Uve1p recognizes non-UV-induced DNA damage, such as platinum-DNA GG diadducts, uracil, dihydrouracil and abasic sites. This is the first time that a single DNA repair endonuclease with the ability to recognize such a diverse range of lesions has been described. This study suggests that Uve1p and the alternative excision repair pathway may participate broadly in the repair of DNA damage.     

Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III

A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae). The enzyme has been purified by a series of column chromatography steps and cleaves OsO4-damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts. The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates. Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations. The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants.     

Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes.

We have previously characterized an extracellular nuclease from Pseudomonas BAL 31 which, in addition to other activities, displays a double-strand exonuclease activity which progressively shortens both strands of linear duplex DNA molecules from both termini. This degradation is accomplished without the introduction of detectable scissions away from the ends of the duplexes. When this nuclease is used to produce a series of progressively shortened samples from a linear duplex DNA, subsequent digestion of these samples with a site-specific restriction endonuclease and analysis of the resulting fragments by gel electrophoresis permits the rapid establishment of the order of the restriction enzyme fragments through the entire genome. This is accomplished by noting from the electropherograms the order in which the various restriction enzyme fragments become noticeably shortened or disappear. Using this method, the five cleavage sites for the endonuclease Hpa I and the single cleavage sites for the nucleases Hpa II and Pst I have been mapped in PM2 bacteriophage DNA. In a more stringent test of the method, 18 of the 24 fragments produced by cleavage of coliphage lambdab2b5c DNA with the Pst I nuclease have been mapped, and five of the six remaining fragments have been assigned to small regions of the genome.     

DNA structure specificity of Rap endonuclease.

The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.     

A new ATP-independent DNA endonuclease from Schizosaccharomyces pombe that recognizes cyclobutane pyrimidine dimers and 6-4 photoproducts.

We have discovered a new DNA endonuclease in the fission yeast Schizosaccharomyces pombe which recognizes cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts. S. pombe DNA endonuclease (SPDE) catalyzes a single ATP-independent incision immediately 5' to the UV photoproduct and generates termini containing 3' hydroxyl and 5' phosphoryl groups. Based on these properties, we propose that SPDE may function in a DNA repair capacity, representing the initial recognition/cleavage step of a DNA excision repair pathway.     

Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E. coli Vsr protein, and human nucleotide excision repair factor XPA

Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones. The understanding of this repair mechanism is a central subject in cell biology. This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins. The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation. The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition. The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence. The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur. The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex. This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA. The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR. This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.     

Cae I: an endonuclease isolated from the African green monkey with properties indicating site-specific cleavage of homologous and heterologous mammalian DNA.

Component alpha DNA is a highly repetitive sequence that comprises nearly a quarter of the African green monkey (Cercopithecus aethiops) genome. A previous microbial restriction enzyme analysis showed that the repeat structure of component alpha DNA is based upon a monomeric unit of 176 +/- 4 base-pairs. An endonuclease, provisionally termed Case I, has been isolated from African green monkey testes that cleaves component alpha DNA into multimeric segments based upon the same repeat periodicity as that revealed by microbial restriction enzymes. The primary sites of Cae I cleavage in the component alpha sequence appear to be 120 +/- 6 base-pairs distant from the Hind III sites and 73 +/- 6 base-pairs distant from the Eco RI* sites. Cae I has been partially characterized with special reference to the effects of ATP and S-adenosylmethionine on the cleavage of component alpha DNA. Cae I may be a member of a class of similar site-specific nucleases present in mammalian cells. Cae I also cleaves mouse satellite DNA into a multimeric series of discrete segments: the periodicity of this series is shorter than that revealed by Eco RII retriction analysis of mouse satellite DNA.     

Domain organization and metal ion requirement of the Type IIS restriction endonuclease MnlI

A two-domain structure of the Type IIS restriction endonuclease MnlI has been identified by limited proteolysis. An N-terminal domain of the enzyme mediates the sequence-specific interaction with DNA, whereas a monomeric C-terminal domain resembles bacterial colicin nucleases in its requirement for alkaline earth as well as transition metal ions for double- and single-stranded DNA cleavage activities. The results indicate that the fusion of the non-specific HNH-type nuclease to the DNA binding domain had transformed MnlI into a Mg(2+)-, Ni(2+)-, Co(2+)-, Mn(2+)-, Zn(2+)-, Ca(2+)-dependent sequence-specific enzyme. Nevertheless, MnlI retains a residual single-stranded DNA cleavage activity controlled by its C-terminal colicin-like nuclease domain.     

Purification and characterization of HeLa endonuclease R. A G-specific mammalian endonuclease

We previously reported a double-stranded endonuclease from HeLa cells, endonuclease R (endo R), which specifically cleaves duplex DNA at sites rich in G.C base pairs. In this report we describe the purification of endo R to near homogeneity by conventional and affinity chromatography. The molecular mass of the active form of endo R is approximately 115-125 kDa. SDS-gel electrophoresis reveals a major protein species of 100 kDa. The enzyme requires Mg2+ as a cofactor and is equally active on closed circular and linear duplex DNA substrates that contain G-rich sequences. A 50% reduction in cleavage activity is observed with Ca2+ ions and no double-stranded cleavage occurs with Zn2+. Use of Mn2+ causes an altered specificity at low concentrations of enzyme or divalent metal ion and nonspecific degradation of the substrate at higher concentrations. Endo R is strongly inhibited by sodium or potassium chloride and exhibits a wide pH optimum of 6.0-9.0. The pI of the enzyme is between 6.5 and 7.0. A 2-fold stimulation is observed with the addition of dGTP or dATP but specific cleavage is inhibited by ATP at an equivalent concentration. Cleavage activity is competitively inhibited 10-fold more efficiently by single-stranded poly(dG)12 than by other DNA competitors. The ends of endo R cleavage products contain 5'-phosphate and 3'-hydroxyl groups, and a significant portion of these products were substrates for T4 DNA ligase. Endo R appears to be a previously uncharacterized mammalian endonuclease.     

Incision at nucleotide insertions/deletions and base pair mismatches by the SP nuclease of spinach

Spinach leaves contain a highly active nuclease called SP. The purified enzyme incises single-stranded DNA, RNA, and double-stranded DNA that has been destabilized by A-T-rich regions and DNA lesions [Strickland et al. (1991) Biochemistry 30, 9749-9756]. This broad range of activity has suggested that SP may be similar to a family of nucleases represented by S1, P1, and the mung bean nuclease. However, unlike these single-stranded nucleases that require acidic pH and low ionic strength conditions, SP has a neutral pH optimum and is active over a wide range of salt concentrations. We have extended these findings and showed that an outstanding substrate for SP is a mismatched DNA duplex. For base-substitution mismatches, SP incises at all mismatches except those containing a guanine residue. SP also cuts at insertion/deletions of one or more nucleotides. Where the extrahelical DNA loop contains one nucleotide, the preference of extrahelical nucleotide is A > T approximately C but undetectable at G. The inability of SP to cut at guanine residues and the favoring of A-T-rich regions distinguish SP from the CEL I family of neutral pH mismatch endonucleases recently discovered in celery and other plants [Oleykowski et al. (1998) Nucleic Acids Res. 26, 4597-4602]. SP, like CEL I, does not turn over after incision at a mismatched site in vitro. Similar to CEL I, the presence of a DNA polymerase or a DNA ligase allows SP to turn over and stimulate its activity in vitro by about 20-fold. The possibility that the SP nuclease may be a natural variant of the CEL I family of mismatch endonucleases is discussed.     

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.     

Single-strand DNA cleavages by eukaryotic topoisomerase II

A new purification method for eukaryotic type II DNA topoisomerase (EC 5.99.1.3) is described, and the avian enzyme has been purified and characterized. An analysis of the cleavage reaction has revealed that topoisomerase II can be trapped as a DNA-enzyme covalent complex containing DNA with double-stranded and single-stranded breaks. The data indicate that DNA cleavage by topoisomerase II proceeds by two asymmetric single-stranded cleavage and resealing steps on opposite strands (separated by 4 bp) with independent probabilities of being trapped upon addition of a protein denaturant. Single-strand cleavages were directly demonstrated at both strong and weak topoisomerase II sites. Thus, a match to the vertebrate topoisomerase II consensus sequence (sequence; see text) (N is any base, and cleavage occurs between -1 and +1) [Spitzner, J.R., & Muller, M.T. (1988) Nucleic Acids Res. 16, 5533-5556)] does not predict whether a cleavage site will be single stranded or double stranded; however, sites cleaved by topoisomerase II that contain two conserved consensus bases (G residue at +2 and T at +4) generally yield double-strand cleavage whereas recognition sites lacking these two consensus elements yield single-strand cleavages. Finally, single-strand cleavages with topoisomerase II do not appear to be an artifact caused by damaged enzyme molecules since topoisomerase II in freshly prepared, crude extracts also shows the property of single-strand cleavages.