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PubMed1997

Processive proofreading by the adenovirus DNA polymerase. Association with the priming protein reduces exonucleolytic degradation.

By using a baculovirus expression system, the adenovirus (Ad) DNA polymerase was purified to homogeneity and shown to display a 3'-->5'exonuclease activity which is coupled to the polymerase activity. On a partial duplex structure the exonuclease activity had a marked preference for excision of a mismatched versus a matched 3'-terminus, which enables the Ad DNA polymerase to act as a proofreading enzyme. On single-stranded DNA the exonuclease action is distributive, but during replication removal of mismatched nucleotides and the switch to synthesis occurs without dissociation of the polymerase from the template. When the Ad DNA polymerase is bound to the precursor terminal protein, the rate of exonucleolysis was four times slower. Moreover, degradation could not proceed as far as with the free Ad polymerase, indicating also a qualitative difference. These results suggest a reduced proofreading capacity of the precursor terminal protein-polymerase complex, which might affect the initial stages of DNA replication.

Adenoviridae↗
PubMed1998

Exonuclease IX of Escherichia coli.

The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA and in both a 5'-->3' and 3'-->5' direction. These enzymes are involved in replicative, repair and recombination functions. We have identified a new exonuclease found in E.coli, termed exonuclease IX, that acts preferentially on single-stranded DNA as a 3'-->5' exonuclease and also functions as a 3'-phosphodiesterase on DNA containing 3'-incised apurinic/apyrimidinic (AP) sites to remove the product trans -4-hydroxy-2-pentenal 5-phosphate. The enzyme showed essentially no activity as a deoxyribophosphodiesterase acting on 5'-incised AP sites. The activity was isolated as a glutathione S-transferase fusion protein from a sequence of the E.coli genome that was 60% identical to a 260 bp region of the small fragment of the DNA polymerase I gene. The protein has a molecular weight of 28 kDa and is free of AP endonuclease and phosphatase activities. Exonuclease IX is expressed in E.coli , as demonstrated by reverse transcription-PCR, and it may function in the DNA base excision repair and other pathways.

DNA, Single-Stranded↗
PubMed1998

Mutational analysis of the 3'-->5' proofreading exonuclease of Escherichia coli DNA polymerase III.

The epsilon subunit of Escherichia coli DNA polymerase III holoenzyme, the enzyme primarily responsible for the duplication of the bacterial chromosome, is a 3'-->5' exonuclease that functions as a proofreader for polymerase errors. In addition, it plays an important structural role within the pol III core. To gain further insight into how epsilon performs these joint structural and catalytic functions, we have investigated a set of 20 newly isolated dnaQ mutator mutants. The mutator effects ranged from strong (700-8000-fold enhancement) to moderate (6-20-fold enhancement), reflecting the range of proofreading deficiencies. Complementation assays revealed most mutators to be partially or fully dominant, suggesting that they carried an exonucleolytic defect but retained binding to the pol III core subunits. One allele, containing a stop codon 3 amino acids from the C-terminal end of the protein, was fully recessive. Sequence analysis of the mutants revealed mutations in the Exo I, Exo II and recently proposed Exo IIIepsilon motifs, as well as in the intervening regions. Together, the data support the functional significance of the proposed motifs, presumably in catalysis, and suggest that the C-terminus of straightepsilon may be specifically involved in binding to the alpha (polymerase) subunit.

Amino Acid Sequence↗
PubMed1998

A stimulatory RNA associated with RecBCD enzyme.

RecBCD enzyme acts in the major pathway of homologous recombination of linear DNA in Escherichia coli. The enzyme unwinds DNA and is an ATP-dependent double-strand and single-strand exonuclease and a single-strand endonuclease; it acts at Chi recombination hotspots (5'-GCTGGTGG-3') to produce a recombinogenic single-stranded DNA 3'-end. We found that a small RNA with a unique sequence of approximately 24 nt was tightly bound to RecBCD enzyme and co-purified with it. When added to native enzyme this RNA, but not four others, increased DNA unwinding and Chi nicking activities of the enzyme. In seven similarly active enzyme preparations the molar ratio of RNA molecules to RecBCD enzyme molecules ranged from 0.2 to <0.008. These results suggest that, although this unique RNA is not an essential enzyme subunit, it has a biological role in stimulating RecBCD enzyme activity.

DNA Helicases↗
PubMed1999

Werner syndrome helicase contains a 5'-->3' exonuclease activity that digests DNA and RNA strands in DNA/DNA and RNA/DNA duplexes dependent on unwinding.

We show that WRN helicase contains a unique 5'-->3' exonuclease activity in the N-terminal region. Adeletion mutant lacking 231 N-terminal amino acid residues, made in a baculovirus system, did nothave this activity, while it showed ATPase and DNA helicase activities. This exonuclease activity was co-precipitated with the helicase activity using monoclonal antibodies specific to WRN helicase, indicating that it is an integral component with WRN helicase. The exonuclease in WRN helicase does not digest free single-stranded DNA or RNA, but it digests a strand in the duplex DNA or an RNA strand in a RNA/DNA heteroduplex in a 5'-->3' direction dependent on duplex unwinding. The digestion products were identified as 5'-mononucleotides. Our data show that WRN helicase needs a single-stranded 3' overhang region for efficient binding and unwinding of duplex molecules, while blunt-ended or 5' overhang duplex molecules were hardly unwound. These findings suggest that the WRN helicase and integral 5'-->3' exonuclease activities are involved in preventing a hyper-recombination by resolving entangled structures of DNA and RNA/DNA heteroduplexes that may be generated during rep-lication, repair and/or transcription.

Animals↗
PubMed1999

In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis.

We have explored the potential of the Tn 552 in vitro transposition reaction as a genetic tool. The reaction is simple (requiring a single protein component), robust and efficient, readily producing insertions into several percent of target DNA. Most importantly, Tn 552 insertions in vitro appear to be essentially random. Extensive analyses indicate that the transposon exhibits no significant regional or sequence specificity for target DNA and leaves no discernible 'cold' spots devoid of insertions. The utility of the in vitro reaction for DNA sequencing was demonstrated with a cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The nucleotide sequence of the entire operon was determined using 71 independent Tn 552 insertions, which generated over 13.5 kb of unique sequence and simultaneously provided a comprehensive collection of insertion mutants. The relatively short ends of Tn 552 make construction of novel transposons a simple process and we describe several useful derivatives. The data presented suggest that Tn 552 transposition is a valuable addition to the arsenal of tools available for molecular biology and genomics.

DNA Transposable Elements↗
PubMed1999

In vivo oligo(A) insertions in phage MS2: role of Escherichia coli poly(A) polymerase.

Previously we introduced an RNase III site into the genome of RNA phage MS2 by extending a hairpin with a perfect 18 bp long stem. One way in which the phage escaped from being killed by RNase III cleavage was to incorporate uncoded A residues on either side of the stem. This oligo(A) stretch interrupts the perfect stem that forms the RNase III site and thus confers resistance. In this paper we have analyzed the origin of these uncoded adenosines. The data strongly suggest that they are added by the host enzyme poly(A) polymerase. Apparently the 3'-OH created by RNase III cleavage becomes a substrate for poly(A) polymerase. Subsequently, MS2 replicase makes one contiguous copy from the two parts of the genome RNA. The evolutionary conversion from RNase III sensitivity to resistance provides a large spectrum of solutions that could be an important tool to understand what essentially constitutes an RNase III site in vivo.

Adenine Nucleotides↗
PubMed1999

Isolation and characterization of the C-terminal nuclease domain from the RecB protein of Escherichia coli.

The RecB subunit of the Escherichia coli RecBCD enzyme has been shown in previous work to have two domains: an N-terminal 100 kDa domain with ATP-dependent helicase activity, and a C-terminal 30 kDa domain. The 30 kDa domain had nuclease activity when linked to a heterologous DNA binding protein, but by itself it appeared unable to bind DNA and lacked detectable nuclease activity. We have expressed and isolated this 30 kDa domain, called RecB(N), and show that it does have nuclease activity detectable at high protein concentration in the presence of polyethylene glycol, added as a molecular crowding agent. The activity is undetectable in a mutant RecB(N)protein in which an aspartate residue has been changed to alanine. Structural analysis of the wild-type and mutant RecB(N)proteins by second derivative absorbance and circular dichroism spectroscopy indicates that both are folded proteins with very similar secondary and tertiary structures. The results show that the Asp-->Ala mutation has not caused a significant structural change in the isolated domain and they support the conclusion that the C-terminal domain of RecB has the sole nuclease active site of RecBCD.

Amino Acid Sequence↗
PubMed1999

3'-Terminal RNA structures and poly(U) tracts inhibit initiation by a 3'-->5' exonuclease in vitro.

We have previously shown that the presence of a poly(A) tail blocks the activity of a highly efficient 3'-->5' exonuclease in HeLa extracts. Similar activities have been implicated in RNA turnover in vivo. It is not clear, however, what protects poly(A)-non-mRNAs from the action of this enzyme. A stem-loop structure located at the 3'-end of U11 RNA was required to protect this transcript from the exonuclease in vitro. Similar 3' stem-loop structures, or extensive base pairinginvolving the 3'-end, are present on all mature small stable RNAs. The placement of artificial stem-loop structures at the 3'-end also protected RNA substrates, suggesting that RNA structure alone is sufficient to block the initiation of the exonuclease. The placement of RNA structures at internal positions of substrate trans-cripts did not affect the activity of the exonuclease or lead to the accumulation of degradation intermediates. Pol III precursor transcripts contain short poly(U) tracts rather than structure at their 3'-ends. Terminal poly(U) tracts protected RNA substrates from the 3'-->5' exonuclease in a protein-dependent fashion. Although La protein is found associated with the terminal U tracts of pol III precursor transcripts both in vivo and in vitro, La protein was not required for poly(U) to protect RNA substrates from the 3'-->5' exonuclease. In summary, these data reveal a variety of ways RNAs have evolved to protect themselves from this exonuclease.

3' Untranslated Regions↗
PubMed1999

Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.

Apurinic Acid↗
PubMed1999

Gene replacement with linear DNA in electroporated wild-type Escherichia coli.

Gene replacement using linear double-stranded DNA fragments in wild-type Escherichia coli transformation is generally inefficient due to exonucleolytic degradation of incoming DNA. Recombination-proficient strains, in which the exonucleolytic activity of RecBCD is inactivated, have been used as transformation recipients to overcome this difficulty. Here we report that gene replacements using linear double-stranded donor DNA can be achieved in wild-type E.coli if electrocompetent cells are used. Using a plasmid target, we obtained 10(2)-10(3) gene replacement events/microgram linear DNA. Using an independent chromosomal target, approximately 60 gene replacement events/microgram linear DNA were obtained. The presence of Chi sites on the linear DNA, which are known to block DNA degradation and stimulate recombination in E.coli, had no effect on gene replacement efficiency in either case. RecBCD-mediated exonucleolytic activity was found to be diminished in electroporated cells. Electrotransformation thus provides a simple way to perform gene replacements in many E.coli strains.

Bacteriophage T4↗
PubMed1999

Synthesis of 5-substituted 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates, their incorporationinto DNA and effects on exonuclease.

Direct PCR sequencing with boronated nucleotides provides an alternative to current PCR sequencing methods. The positions of boranophosphate-modified nucleotides incorporated randomly into DNA during PCR can be revealed directly by exonuclease digestion to give sequencing ladders. Cytosine nucleotides, however, are especially sensitive to exonuclease digestion and provide suboptimal sequencing ladders. Therefore, a series of 5-substituted analogs of 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates (dCTPalphaB) were synthesized with the hope of increasing the nuclease resistance of deoxycytosine residues and thereby enhancing the deoxycytosine band intensities. These dCTP analogs contain a boranophosphate modification at the alpha-phosphate group in 2'-deoxycytidine 5'-triphosphate (dCTP) as well as a 5-methyl, 5-ethyl, 5-bromo or 5-iodo substitution for the 5-hydrogen of cytosine. The two diastereomers of each new dCTP derivative were separated by reverse phase HPLC. The first eluted diastereomer (putatively Rp) of each dCTP analog was a substrate for T7 DNA polymerase (Sequenase) and had an incorporation efficiency similar to normal dCTP and dCTPalphaB, with the 5-iodo-dCTPalphaB analog being the least efficient. Substitution at the C-5 position of cytosine by alkyl groups (ethyl and methyl) markedly enhanced the dCTPalphaB resistance towards exonuclease III (5-Et-dCTPalphaB >5-Me-dCTPalphaB >dCTPalphaB approximately 5-Br-dCTPalphaB >5-I-dCTPalphaB), thereby generating DNA sequences that better define the deoxycytosine positions. The introduction of modified dCTPalphaB should increase the utility of direct DNA sequencing with boronated nucleoside 5'-triphosphates.

Boron Compounds↗
PubMed2000

Characterization of the human and mouse WRN 3'-->5' exonuclease.

Werner's syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of a partial array of age-associated pathologies. WRN, the gene defective in WS, encodes a 1432 amino acid protein (hWRN) with intrinsic 3'-->5' DNA helicase activity. We recently showed that hWRN is also a 3'-->5' exonuclease. Here, we further characterize the hWRN exonuclease. hWRN efficiently degraded the 3' recessed strands of double-stranded DNA or a DNA-RNA heteroduplex. It had little or no activity on blunt-ended DNA, DNA with a 3' protruding strand, or single-stranded DNA. The hWRN exonuclease efficiently removed a mismatched nucleotide at a 3' recessed terminus, and was capable of initiating DNA degradation from a 12-nt gap, or a nick. We further show that the mouse WRN (mWRN) is also a 3'-->5' exonuclease, with substrate specificity similar to that of hWRN. Finally, we show that hWRN forms a trimer and interacts with the proliferating cell nuclear antigen in vitro. These findings provide new data on the biochemical activities of WRN that may help elucidate its role(s) in DNA metabolism.

Amino Acid Sequence↗
PubMed2000

The nature of the 5'-terminus is a major determinant for DNA processing by Schizosaccharomyces pombe Rad2p, a FEN-1 family nuclease.

The nuclease activity of FEN-1 is essential for both DNA replication and repair. Intermediate DNA products formed during these processes possess a variety of structures and termini. We have previously demonstrated that the 5'-->3' exonuclease activity of the Schizosaccharomyces pombe FEN-1 protein Rad2p requires a 5'-phosphoryl moiety to efficiently degrade a nick-containing substrate in a reconstituted alternative excision repair system. Here we report the effect of different 5'-terminal moieties of a variety of DNA substrates on Rad2p activity. We also show that Rad2p possesses a 5'-->3' single-stranded exonuclease activity, similar to Saccharomyces cerevisiae Rad27p and phage T5 5'-->3' exonuclease (also a FEN-1 homolog). FEN-1 nucleases have been associated with the base excision repair pathway, specifically processing cleaved abasic sites. Because several enzymes cleave abasic sites through different mechanisms resulting in different 5'-termini, we investigated the ability of Rad2p to process several different types of cleaved abasic sites. With varying efficiency, Rad2p degrades the products of an abasic site cleaved by Escherichia coli endonuclease III and endonuclease IV (prototype AP endonucleases) and S.POMBE: Uve1p. These results provide important insights into the roles of Rad2p in DNA repair processes in S.POMBE:

Carbon-Oxygen Lyases↗
PubMed2002

Molecular interactions of human Exo1 with DNA.

Human Exo1 is a member of the RAD2 nuclease family with roles in replication, repair and recombination. Despite sharing significant amino acid sequence homology, the RAD2 proteins exhibit disparate nuclease properties and biological functions. In order to identify elements that dictate substrate selectivity within the RAD2 family, we sought to identify residues key to Exo1 nuclease activity and to characterize the molecular details of the human Exo1-DNA interaction. Site-specific mutagenesis studies demonstrate that amino acids D78, D173 and D225 are critical for Exo1 nuclease function. In addition, we show that the chemical nature of the 5'-terminus has a major impact on Exo1 nuclease efficiency, with a 5'-phosphate group stimulating degradation 10-fold and a 5'-biotin inhibiting degradation 10-fold (relative to a 5'-hydroxyl moiety). An abasic lesion located within a substrate DNA strand impedes Exo1 nucleolytic degradation, and a 5'-terminal abasic residue reduces nuclease efficiency 2-fold. Hydroxyl radical footprinting indicates that Exo1 binds predominantly along the minor groove of flap DNA, downstream of the junction. As will be discussed, our results favor the notion that the single-stranded DNA structure is pinched by the helical arch of the protein and not threaded through this key recognition loop. Furthermore, our studies indicate that significant, presumably biologically relevant, differences exist between the active site dynamics of Exo1 and Fen1.

Base Sequence↗
PubMed2002

Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H2O2.

The structure-specific FEN-1 endonuclease has been implicated in various cellular processes, including DNA replication, repair and recombination. In vertebrate cells, however, no in vivo evidence has been provided so far. Here, we knocked out the FEN-1 gene (FEN1) in the chicken DT40 cell line. Surprisingly, homozygous mutant (FEN1-/-) cells were viable, indicating that FEN-1 is not essential for cell proliferation and thus for Okazaki fragment processing during DNA replication. However, compared with wild-type cells, FEN1-/- cells exhibited a slow growth phenotype, probably due to a high rate of cell death. The mutant cells were hypersensitive to methylmethane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and H2O2, but not to UV light, X-rays and etoposide, suggesting that FEN-1 functions in base excision repair in vertebrate cells.

Animals↗
PubMed1990

Coprecipitation of 3'----5' exonuclease and DNA polymerase gamma with anti-DNA polymerase gamma antibody.

Highly purified preparations of chick embryo DNA polymerase gamma contained 3'----5' exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T.A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase gamma precipitated 3'----5' exonuclease activity to the same extent as DNA polymerase gamma activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase gamma activity to thermal treatment at 50 degrees C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase gamma and 3'----5' exonuclease are, although not identical, closely correlated.

Animals↗
PubMed1982

Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione.

ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography. Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion. The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction. The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine. These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.

Arginine↗