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PubMed1990

Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. I. Purification and some properties of AP DNA endonucleases in rat liver chromatin.

Three kinds of apurinic/apyrimidinic (AP) DNA endonuclease, APcI, APcII, APcIII, were purified from rat liver chromatin through 1M KCl extraction, DEAE-trisacryl ion exchange chromatography. Sephadex G-150 gel filtration and AP DNA cellulose affinity chromatography. Activities of the purified APcI, APcII and APcIII were 62.5, 83.3 and 52.0 EU/mg of protein, respectively. Molecular weights of APcI, APcII and APcIII, each consisting of a single polypeptide, were 30,000, 42,000 and 13,000, and isoelectric points of them were 7.2, 6.3 and 6.2, respectively. Three enzymes showed different substrate specificities; APcI acted only on AP DNA, and APcII acted on both AP DNA and UV DNA, while APcIII acted on 3'-methyl-4-monomethylaminoazobenzene (3'-Me MAB) DNA adduct as well as AP DNA and UV DNA. These results indicate that three kinds of AP DNA endonuclease present in rat liver chromatin have structural and functional diversities.

Animals↗
PubMed1990

Substrate specificity of HeLa endonuclease R. A G-specific mammalian endonuclease.

We examined the substrate specificity of endonuclease R (endo R) a mammalian endonuclease that cleaves G.C-rich DNA sequences. The best substrates for double-stranded cleavage were homopolymeric stretches of poly(dG).poly(dC). Plasmids which contain other G-rich sequences were also cleaved but at a reduced frequency. These included the telomeric sequences, d(G4T2) and d(G2-6A), which were cleaved at approximately one-third the frequency of d(G)n.d(C)n. The alternating copolymer d(GA) and the terminal sequences of adeno-associated virus d(G1-3T/A) were also cut. Poly(dA).poly(dT) and the alternating copolymer d(GC)n were not detectably cleaved. Although endo R has a nicking activity which converts supercoiled plasmids to nicked circular DNA, the nicking activity is random with respect to plasmid sequences. Specific cleavage of G-rich sequences appears to occur by a concerted double-stranded mechanism. The cleavage pattern within the G-rich runs suggests that cleavage can occur anywhere within the G-rich region. Product ligation experiments indicate that a limited number of cleavage events (1-2) occur/molecule. Inasmuch as the best substrates for endo R are d(G)n.d(C)n and telomeric sequences, we suggest that endo R may directly recognize and cleave DNA that contains G.G base pairing.

Base Composition↗
PubMed1980

Restriction endonucleases or the site-specific DNA endonucleases.

Our present view of the site-specific endonucleases, which appear to be ubiquituous in the prokaryote kingdom, is probably heavily distorted by our search for tools for recombinant DNA technology. Only those enzymes having recognition sequences in the range of three to seven specific bases have been isolated. Of course the usefulness of these enzymes in the analysis of complex genomes, the rise of "reverse genetics", and the immediate breakthroughs in the area of gene expression in eukaryotes, particularly the understanding of tumour virus RNA processing and gene rearrangements in the expression of immunoglobulin genes has dominated the consciousness of the molecular and cell biologists during the last five years. There is a great diversity of staggering, symmetry, asymmetry and degeneration in the recognition sequences found. Taking into account also the genetic data on site-specific recombination and/or DNA degradation suggests that our present collection of endonucleases may only represent a narrow spectrum of specificities on an open-ended scale of complexity. The enzymes, themselves provide a rich pool to be exploited by the biophysicist and the biochemist to probe the subtleties of DNA-protein interaction.

Binding Sites↗
PubMedDate not supplied

Stimulation of human flap endonuclease 1 by human immunodeficiency virus type 1 integrase: possible role for flap endonuclease 1 in 5'-end processing of human immunodeficiency virus type 1 integration intermediates.

Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5'-AC unpaired dinucleotide. These short-flap (pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5'-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5'-flaps from short- and long-flap oligonucleotide substrates that mimic pre- and post-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5'-flaps by FEN-1 in pre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5'-flaps by FEN-1 from post-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5'-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen with pre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall, post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase than pre-repair structures. The site of cleavage was 1 or 2 nucleotides 3' of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5'-flaps from either pre- or post-repair substrates.

DNA Repair↗
PubMed2000

Ultraviolet damage endonuclease (Uve1p): a structure and strand-specific DNA endonuclease.

Schizosaccharomyces pombe ultraviolet damage endonuclease (UVDE or Uve1p) performs the initial step in an alternative excision repair pathway for UV-induced DNA damage. This DNA repair pathway was originally thought to be specific for UV damage. However, the broad substrate specificity of Uve1p suggests a more general role for this enzyme. Uve1p recognizes UV-induced bipyrimidine photoadducts and other non-UV-induced DNA adducts. Biochemical and genetic analysis also suggests that Uve1p may be involved in orchestrating mismatch repair in vivo. This study demonstrates that Uve1p recognizes and cleaves heteroduplex DNA with small unpaired loops but does not recognize loops six to eight nucleotides in length. In addition, the enzyme does not recognize DNA with palindromic insertions that could form base-paired hairpin structures. The cleavage efficiency of Uve1p depends on the distance of a mismatch from the DNA terminus, suggesting that the 3' terminus may contribute to the strand discrimination signal for Uve1p. These biochemical activities are discussed in the context of the role of Uve1p in DNA repair.

Base Pair Mismatch↗
PubMed1996

Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.

DNA↗
PubMed2000

The genes encoding endonuclease VIII and endonuclease III in Escherichia coli are transcribed as the terminal genes in operons.

Escherichia coli endonuclease VIII and endo-nuclease III are oxidative base excision repair DNA glycosylases that remove oxidized pyrimidines from DNA. The genes encoding these proteins, nei and nth, are both co-transcribed as the terminal genes in operons. nei is the terminal gene in an operon with four open reading frames that encode proteins of unknown function. This operon has two confirmed transcription initiation sites upstream of the first open reading frame and two transcript termination sites downstream of nei. nth is the terminal gene in an operon with seven open reading frames that encode proteins of unknown function. The six open reading frames immediately upstream of nth show homology to the genes rnfA, rnfB, rnfC, rnfD, rnfG and rnfE from Rhodobacter capsulatis. The rnf genes are required for nitrogen fixation in R.capsulatis and have been predicted to make up a membrane complex involved in electron transport to nitrogenase. The nth operon has transcription initiation sites upstream of the first and second open reading frames and a single transcript termination site downstream of nth. The order of genes in these operons has been conserved or partially conserved in other bacteria, although it is not known whether the genes are co-transcribed in these other organisms.

Conserved Sequence↗
PubMed2002

Human AP-endonuclease 1 and hnRNP-L interact with a nCaRE-like repressor element in the AP-endonuclease 1 promoter.

The major human AP-endonuclease 1 (APE1) is a multifunctional protein that plays a central role in the repair of damaged DNA by acting as a dual-function nuclease in the base excision repair pathway. This enzyme was also independently identified as a redox activator of AP-1 DNA-binding activity and has subsequently been shown to activate a variety of transcription factors via a redox mechanism. In a third distinct role, APE1 was identified as a component of a trans-acting complex that acts as a repressor by binding to the negative calcium responsive elements (nCaRE)-A and nCaRE-B, which were first discovered in the promoter of the human parathyroid gene and later in the APE1 promoter itself. Here we show that the nuclear protein complex which binds to the nCaRE-B2 of the hAPE1 gene contains APE1 itself and the heterogeneous nuclear ribonucleoprotein L (hnRNP-L). The interaction between the APE1 and hnRNP-L proteins does not require the presence of nCaRE-B2. Our results support the possibility that the APE1 gene is down-regulated by its own product, which would be the first such example of the regulation of a DNA repair enzyme, and identify a novel function of hnRNP-L in transcriptional regulation.

Amino Acid Sequence↗
PubMed2006

Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis.

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5' phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5'-dTdCdA-3' but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5'-dTdCdA-3', which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.

Bacteriophage T4↗
PubMed1999

Site-specific endonuclease NspLKI is an isoschizomer of endonuclease HaeIII.

Site-specific endonuclease NspLKI has been isolated and purified to functionally pure state from soil bacterium Nocardia species LK by successive chromatography on columns with phosphocellulose, HTP hydroxyapatite, and heparin-Sepharose. The isolated enzyme recognizes the 5'-GG downward arrowCC-3' sequence on DNA and cleaves it as indicated by the arrow, i.e., it is an isoschizomer of HaeIII. The final enzyme yield is 1.105 units per gram of wet biomass. The enzyme is active in the temperature range of 25-60 degrees C with an optimum at 48-55 degrees C; it does not lose activity on storage for three days at room temperature. An optimal buffer is HRB containing 10 mM Tris-HCl, pH 7.4, 200 microgram/ml albumin, 10 mM MgCl2, and 100 mM NaCl.

Chromatography, Ion Exchange↗
PubMed2004

Alpha-anomeric deoxynucleotides, anoxic products of ionizing radiation, are substrates for the endonuclease IV-type AP endonucleases.

Alpha-anomeric 2'-deoxynucleosides (alphadN) are one of the products formed by ionizing radiation (IR) in DNA under anoxic conditions. Alpha-2'-deoxyadenosine (alphadA) and alpha-thymidine (alphaT) are not recognized by DNA glycosylases, and are likely removed by the alternative nucleotide incision repair (NIR) pathway. Indeed, it has been shown that alphadA is a substrate for the Escherichia coli Nfo and human Ape1 proteins. However, the repair pathway for removal of alphadA and other alphadN in yeast is unknown. Here we report that alphadA when present in DNA is recognized by the Saccharomyces cerevisiae Apn1 protein, a homologue of Nfo. Furthermore, alphaT is a substrate for Nfo and Apn1. Kinetic constants indicate that alphadA and alphaT are equally good substrates, as a tetrahydrofuranyl (THF) residue, for Nfo and Apn1. Using E. coli and S. cerevisiae cell-free extracts, we have further substantiated the role of the nfo and apn1 gene products in the repair of alphadN. Surprisingly, we found that bacteria and yeast NIR-deficient mutants are not sensitive to IR, suggesting that DNA strand breaks with terminal 3'-blocking groups rather than alphadN might contribute to cell survival. We propose that the novel substrate specificities of Nfo and Apn1 play an important role in counteracting oxidative DNA base damage.

Anaerobiosis↗