Properties of a DNA repair endonuclease from mouse plasmacytoma cells

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.     

In vitro synthesis of short DNA pieces by DNA polymerase alpha from mouse myeloma

The DNA chain elongation mechanisms of mouse DNA polymerases alpha and beta have been analyzed by using denatured DNA with a (dT)n block at the 3'-end as a template in combination with RNA ((rA)12-20)primer. The (rA)12-20-primed DNA product synthesized by DNA polymerase alpha was 3-5 s in size even after prolonged reaction; instead of a size increase, the number of 3-5 s molecules increased with the reaction time. The size of products was not affected by differences in 3H-labeled substrate (dATP or dTTP), enzyme amount, KCl concentration, or the length of 3'-(dT)n blocks. On the other hand, DNA polymerase beta synthesized long DNA products by a highly distributive reaction mechanism. 3-5 sDNA pieces synthesized by DNA polymerase alpha were not elongated any further by DNA polymerase alpha, but were converted into long DNA chains by DNA polymerase beta. The results imply that DNA polymerase alpha recognizes the size of the product DNA, and shuts off further elongation.     

Nucleotide sequence of polypyrimidines from cloned mouse DNA as determined by base-specific blockage of exonuclease action

Cloned fragments of mouse DNA have been screened for the presence of long polypyrimidine/polypurine segments. The polypyrimidine portion of one such segment (about 200 nucleotides in length) has been isolated by acidic depurination of the entire cloned fragment and plasmid vector followed by selective precipitation and 5'-32P labeling. This polypyrimidine has been used to demonstrate a new procedure for sequencing. Covalent modification of thymine with a water-soluble carbodiimide, or cytosine with glutaric anhydride, at low levels blocked the action of snake venom exonuclease. After deblocking, separation of the products of digestion by polyacrylamide gel electrophoresis yields a sequence ladder which can be used to determine the position of C and T residues as in other sequencing methods. A sequence of 72 residues adjacent to the 5' end has been established, consisting principally of the repeating tetranucleotide (CCTT)n. A low ratio of endonuclease to exonuclease is essential for application of this method to sequences of this size. Accordingly, a very sensitive modification of a fluorometric endonuclease assay was developed and used to optimize pH and Mg2+ conditions to favor exonuclease activity over the accompanying endonuclease activity. The results clearly indicate that long polypyrimidine tracts can be efficiently prepared and their sequences determined with this method using commercially available exonuclease preparations without additional purification.     

A single-stranded DNA exonuclease from Schizosaccharomyces pombe.

We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe. The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell. It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates. Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair.     

An ATPase depending on the presence of single-stranded DNA from mouse myeloma.

An ATPase was purified from mouse myeloma MOPC 70E the activity of which depends on the presence of single-stranded DNA and divalent cations such as Mg2+, Mn2+, Ca2+, Ni2+ or Fe2+. The enzyme splits both ribonucleoside and deoxyribonucleoside triphosphates but preferentially ATP and dATP yielding nucleoside diphosphates and inorganic phosphate. The enzyme has an absolute requirement for single-stranded DNA. Alternating double-stranded polydeoxynucleotides are only slight effective, and native double-stranded DNA, single-stranded and double-stranded RNAs as well as DNA - RNA hybrids are ineffective in stimulating the ATPase. The enzyme has further characterized by sedimentation in a sucrose density gradient (s20, w = 5.5 S) and by isoelectric focussing in an ampholine pH gradient (pI = 6.5).     

Coexistence of endonuclease and exonuclease activities in a novel RecJ from Bacillus cereus

Biotechnology Letters - All RecJ proteins are known to date only perform exonuclease activity. The present study reports that a novel RecJ protein obtained from Bacillus cereus isolated from marine...     

High-molecular-weight DNA polymerases from mouse myeloma. Purification and properties of three enzymes.

Cytoplasmic (high-molecular-weight) DNA polymerase was partially purified from mouse myeloma. Upon chromatography on DEAE-Sephadex, following fractionation on phosphocellulose, the enzyme was resolved into three species named CI, CII, and CIII. The species CI and CII have equal sedimentation coefficients (10.5 S) in sucrose gradients without salt. In the presence of 125 mM ammonium sulfate the sedimentation coefficients are reduced to 8.6 S. The species CIII shows sedimentation coefficients of 5.7 S and 5.2 S without salt and in the presence of 125 mM ammonium sulfate, respectively. This species is assumed to be an artifact arising from either CI or to a minor extent from CII. The optima for pH, KCl and Mg2+ concentration, and the extent of inhibition by N-ethylmaleimide are the same. However, the enzymes differ in their responses to Mn2+ (substituting for Mg-2+), and to addition of ethanol, dimethylsulfoxide, and various phospholipids in the assay mixture. The enzymes prefer poly[d(A-T - d(A-T)] or partially degraded (activated) DNA as template rather than double-stranded or single-stranded DNA. The activity on activated DNA relative to that on poly[d(A-T) - D(A-T)] was found to be 93, 66, and 29% for DNA polymerases CI, CII, and CIII, respectively.     

Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions 50-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Bal31 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M13 mp10 or mp11. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.     

Adenovirus terminal protein protects single stranded DNA from digestion by a cellular exonuclease.

Adenovirus 5 DNA-protein complex is isolated from virions as a duplex DNA molecule covalently attached by the 5' termini of each strand to virion protein of unknown function. The DNA-protein complex can be digested with E. coli exonuclease III to generate molecules analogous to DNA replication intermediates in that they contain long single stranded regions ending in 5' termini bound to terminal protein. The infectivity of pronase digested Adenovirus 5 DNA is greatly diminished by exonuclease III digestion. However, the infectivity of the DNA-protein complex is not significantly altered when up to at least 2400 nucleotides are removed from the 3' ends of each strand. This indicates that the terminal protein protects 5' terminated single stranded regions from digestion by a cellular exonuclease. DNA-protein complex prepared from a host range mutant with a mutation mapping in the left 4% of the genome was digested with exonuclease III, hybridized to a wild type restriction fragment comprising the left 8% of the genome, and transfected into HeLa cells. Virus with wild type phenotype was recovered at high frequency.     

Properties of herpes simplex virus DNA polymerase and characterization of its associated exonuclease activity.

Herpes simplex virus (HSV) DNA polymerase was isolated on a large-scale from African green monkey kidney cells infected with HSV type 1 (HSV-1) strain Angelotti. After DNA-cellulose chromatography the enzyme showed a specific activity of 48,000 units/mg protein. Three major single polypeptides with molecular weights of 144,000, 74,000 and 29,000 were copurified with the enzyme activity at the DNA-cellulose ste. By its chromatographic behavior and by template studies, the HSV DNA polymerase activity was clearly distinguishable from cellular alpha, beta and gamma DNA polymerase activities. Two exonucleolytic activities were found in the DNA-cellulose enzyme preparation. The main exonucleolytic activity, which degraded both single-stranded and double-stranded DNA to deoxynucleoside 5'-monophosphates, was separated by subsequent velocity sedimentation. The remaining exonucleolytic activity was not separable from the HSV DNA polymerase by several chromatographic steps and by velocity sedimentation at high ionic strength. This novel exonuclease and HSV DNA polymerase were equally sensitive both to phosphonoacetic acid and Zn2+ ions, inhibitors of the viral polymerase. Similar to the 3'-to-5'-exonuclease of procaryotic DNA polymerases and mammalian DNA polymerase delta, the HSV-polymerase-associated exonuclease catalyzed the removal of 3'-terminal nucleotides from the primer/template as well as the template-dependent conversion of deoxynucleoside triphosphates to monophosphates.     

The novel function of a short region K253 XRXXXD259 conserved in the exonuclease domain of hyperthermostable DNA polymerase I from Pyrococcus horikoshii

The DNA polymerase gene of the hyperthermophile Pyrococcus horikoshii was successfully overexpressed after removing an intein. The importance of an amino acid sequence around a highly conserved Asp was studied by site-directed mutagenesis. The results indicated that Lys253, Arg255, and Asp259 form a novel functional motif, K253xRxxxD259 (outside known motifs Exo I, II, and III), that is important not only for exonuclease activity but also for polymerizing activity, confirming functional interdependence between the polymerase and exonuclease domains. The short loop region, K253G254R255, probably contributes to binding to DNA substrates. Moreover, the negative charge and the side-chain length of D259 might play a supporting role in coordinating the conserved Mg2+ to the correct position at the active center in the exonuclease domain.     

Stereochemical course of hydrolysis of DNA by exonuclease I from Escherichia coli

Exonuclease I has been purified from an overproducing strain of Escherichia coli K12 [Prasher, D. C., Conarro, L., & Kushner, S. R. (1983) J. Biol. Chem. 258, 6340-6343]. The enzyme hydrolyzes deoxyribonucleic acids that contain chiral phosphorothioate diester linkages, and the stereochemical course of the reaction is inversion of configuration at phosphorus. This result is most consistent with hydrolysis occurring via the direct attack of water on a phosphorothioate diester rather than through the intermediacy of a covalent nucleotidyl-enzyme intermediate. This finding represents the first example of a processive exonuclease whose stereochemical pathway has been determined.     

Properties of the 3' to 5' exonuclease associated with calf DNA polymerase delta

The 3' to 5' exonuclease of calf thymus DNA polymerase delta has properties expected of a proofreading nuclease. It digests either single-stranded DNA or the single-stranded nucleotides of a mismatched primer on a DNA template by a nonprocessive mechanism. The distribution of oligonucleotide products suggests that a significant portion of the enzyme dissociates after the removal of one nucleotide. This mechanism is expected if the substrate in vivo is an incorrect nucleotide added by the polymerase. Digestion of single-stranded DNA does not proceed to completion, producing final products six to seven nucleotides long. Digestion of a long mismatched terminus accelerates when the mismatched region is reduced to less than six nucleotides. At the point of complementation, the digestion rate is greatly reduced. These results suggest that short mismatched regions are a preferred substrate. The use of a mismatched primer-template analogue, lacking the template single strand, greatly lowers digestion efficiency at the single-stranded 3'-terminus, suggesting that the template strand is important for substrate recognition. When oligonucleotides were examined for effectiveness as exonuclease inhibitors, (dG)8 was found to be the most potent inhibitor of single-stranded DNA digestion. (dG)8 was less effective at inhibiting digestion of mismatched primer termini, again suggesting that this DNA is a preferred substrate. Overall, these results indicate that the exonuclease of DNA polymerase delta efficiently removes short mismatched DNA, a structure formed from misincorporation during DNA synthesis.     

枯草芽孢杆菌中一种新型双向启动子的功能鉴定

本研究旨在通过转录组分析预测的方法,由地衣芽孢杆菌中筛选获得一种新型双向启动子,鉴定其启动强度。以已知强组成型启动子pShuttle-09为对照,检测其对克劳氏芽孢杆菌碱性蛋白酶基因的表达活性。成功构建了3种重组碱性蛋白酶表达载体及对应的工程菌株。在新型启动子pLA和其反向启动子pLB调控转录下,克劳氏芽孢杆菌碱性蛋白酶表达活性达到164 U/mL和111 U/mL。结果表明,pLA的启动强度明显高于pShuttle-09和pLB,pLA启动子与pLB启动子均可表达碱性蛋白酶。从而为枯草芽孢杆菌表达系统中异源基因的表达提供一个新的方向,也为原核生物中共同表达两种基因提供了新的思路。     

Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.     

Strand exchange protein 1 from Saccharomyces cerevisiae. A novel multifunctional protein that contains DNA strand exchange and exonuclease activities

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA molecules from single-stranded circles and homologous linear duplex DNA in vitro. Previously, Sep1 was purified as a 132,000-Da species; however, DNA sequence analysis indicates that the SEP1 gene is capable of encoding a 175,000-Da protein (Tishkoff, D.X., Johnson, A.W., and Kolodner, R.D. (1991) Mol. Cell. Biol. 11, 2593-2608). The SEP1 gene was cloned into a GAL10 expression vector and expressed in a protease-deficient yeast strain. Intact Sep1, which migrated as a Mr-160,000 polypeptide during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to apparent homogeneity and shown to have activities similar to those of the originally purified Mr = 132,000 fragment. We report here that, in addition to strand exchange activity, Sep1 contains an intrinsic exonuclease that is active on single- and double-stranded DNA with a severalfold preference for single-stranded DNA. The nuclease was induced in crude extracts upon induction with galactose, it co-purified with the strand exchange activity of Sep1, and the nuclease and strand exchange activities of Sep1 showed the same kinetics of heat inactivation. Sep1 nuclease, which requires Mg2+, can be functionally separated from the strand exchange activity by the substitution of Ca2+ for Mg2+. Under these conditions, the nuclease is inactive, and strand exchange activity is dependent on prior resection of the DNA ends by an exogenous exonuclease. Thus, the nuclease is necessary for synapsis but not strand exchange. Electron microscopic analysis revealed that true strand exchange products, alpha molecules and nicked double-stranded circular molecules, were formed. In addition, strand transfer proceeded to similar extents on 5'-resected and 3'-resected DNA. This result suggests that the polarity of strand transfer by Sep1 is determined by the polarity of its intrinsic nuclease.     

Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA

Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability. The WS protein (WRN) possesses 3′→5′ DNA helicase and associated ATPase activities, as well as 3′→5′ DNA exonuclease activity. Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity. It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype. WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme. We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD. We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis. Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation. We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.