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PubMed1992

Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities.

In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation. In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors. Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process. Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.

Amino Acid Sequence↗
PubMed1993

Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli.

Escherichia coli cells induced for the SOS response express inducible stable DNA replication (iSDR) as an SOS function. Initiation of iSDR is independent of transcription, translation and DnaA protein, which are essential for initiation of DNA replication from oriC. We found that a recA mutant that is defective in recombination but proficient in SOS induction could not elicit iSDR. In contrast, iSDR was enhanced by recD and recJ mutations that inactivate the exonuclease V activity of the RecBCD enzyme and the RecJ exonuclease activity, respectively. A mutation in the ruvC gene that blocks the resolution of recombination intermediates (i.e. Holliday structures) also enhanced iSDR. Furthermore, inhibition of branch migration by recG or ruvAB mutations dramatically increased the iSDR activity. recBC mutants are defective in iSDR induction but the defect was suppressed by a mutation in the sbcA gene. The major product of minichromosomes replicated by iSDR was covalently closed circular monomers. We propose that recombination intermediates (i.e. D-loop structures) created by the action of RecA recombinase and RecBC(D) helicase play a central role in initiation of iSDR.

Bacterial Proteins↗
PubMed1994

Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi-less DNA. Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.

Base Sequence↗
PubMed1986

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Binding Sites↗
PubMed1998

Salt-stable complexes of the Escherichia coli RecBCD enzyme bound to double-stranded DNA.

We have examined binding of the RecBCD enzyme to linear double-stranded DNA under two types of conditions. Binding in the absence of ATP can be measured by a nitrocellulose filter binding assay or by gel retardation on polyacrylamide gels. The binding is tightest to ends with four-nucleotide single-stranded 3'-termini (3'-overhang > 5'-overhang > blunt ends). The Kd for blunt ends is 3. 0 (+/-0.5) nM, in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2. The binding is weakened in the presence of NaCl, with none detected at 0.5 M NaCl. Binding in the presence of ATP and low MgCl2 concentrations ("unwinding conditions") can be measured by the filter assay and on agarose gels. Enzyme-DNA complexes allowed to form under unwinding conditions are not affected by 0.5 M NaCl. ATP hydrolysis continues and the complexes dissociate at a rate similar to those to which no salt is added. These enzyme-DNA complexes can be trapped with EDTA, and they are unaffected for at least 1 h by 0.5 M NaCl or heparin. The results show that the enzyme-DNA interactions are different when the enzyme is bound to partially unwound DNA compared to when it is bound to the ends of fully duplex DNA.

Adenosine Triphosphate↗
PubMed1995

A slot blot method for detection of ultraviolet photoproducts in DNA.

A simple new method was established to detect the major uv photoproducts in DNA. A slot blot method, involving the use of T4 DNA polymerase-associated (3'--> 5') exonuclease digestion of uv-irradiated DNA, was used to detect (primarily) cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) dimers. Hydrolysis of DNA by this enzyme is quantitatively blocked by both of these photoproducts. To detect (primarily) (6-4) dimers, Escherichia coli DNA photolyase was utilized to specifically reverse cyclobutane pyrimidine dimers.

DNA↗
PubMed1996

"Doublex" fluorescent DNA sequencing: two independent sequences obtained simultaneously in one reaction with internal labeling and unlabeled primers.

The novel "doublex" DNA sequencing technique that makes it possible to obtain simultaneously two independent sequences from one sequencing reaction with the use of unlabeled primers and internal labeling is described. The different sequencing products are labeled in parallel with fluorescein-15-dATP and Texas red-5-dCTP present in the same tube. The characteristics of T7 DNA polymerase are exploited to ensure that only either of the labeled dNTPs is incorporated into the corresponding sequencing products. Specificity of labeling is ensured by the selection of primers. One of the unlabeled primers is chosen to be followed by an "A," the other by a "C" to be incorporated immediately downstream from the primer binding site. The doublex sequencing technique is applicable to the simultaneous sequencing of either the same DNA template/strand or a mixture of different templates. Combinations of unlabeled and labeled primers in the same sequencing reaction are also possible. The two sequences can be determined in parallel and on-line in the same lanes of a gel with a novel automated DNA sequencer, which was previously described for use with labeled primers.

Autoanalysis↗
PubMed2002

A strategy for examining complex mixtures of deoxyoligonucleotides using ion-pair-reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and informatics.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ion-pair-reverse-phase high-performance liquid chromatography (IP-RP HPLC) techniques were combined to determine the sequence identity of short single-stranded deoxyoligonucleotides. This methodology is demonstrated using a commercially available multiplex set of eight primer pairs. The primer pairs were separated and collected by IP-RP HPLC. Partial sequence information for IP-RP HPLC fractions was obtained from analyzing exonuclease digestion products by MALDI-TOF MS. IP-RP HPLC, MALDI-TOF MS, exonuclease digests, and a simple computational algorithm provide an integrated strategy for determining the sequence of short nucleic acid oligomers.

Algorithms↗
PubMed1999

Characterization of a 3'-5' exonuclease associated with VDJP.

VDJP (V(D)J RSS Dependent DNA Joining Protein) was cloned based on binding to the nonamer portion of the V(D)J recombinational signal sequence (RSS), and genetic analysis revealed that VDJP is encoded by the same gene as the large subunit of Replication Factor C (RF-C). Recombinant VDJP has a site directed DNA joining activity and is capable of forming a covalent bond between DNA fragments containing an RSS element near their ends and exhibits 3' to 5' exonuclease activity. In this report, we examine the biochemical properties of the VDJP exonuclease activity such as directionality of nuclease action (3' to 5' or 5' to 3'), single-strand substrate preference, cleavage products, dependence on cofactors and metal cations, and optimal reaction conditions. From this analysis, we conclude that VDJP has an intrinsic 3'-5' exonuclease activity that produces mononucleotide products.

DNA↗
PubMed1999

A real time quantitative PCR-based method for the detection and quantification of simian virus 40.

A real time quantitative PCR-based simian virus 40 (SV40) detection and quantification method has been developed. This method takes advantage of the 5' to 3'-exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for direct monitoring of PCR product accumulation through a dual-labelled fluorogenic probe. This method provides accurate, precise and reproducible quantification of SV40 DNA over a linear dynamic range of at least 100,000-fold with a minimum detection level of 6.4 copy equivalents/microL of SV40 viral particle in test samples. The sample preparation procedure employed allows for efficient and consistent recovery of SV40 DNA from test samples. High concentrations of protein and cellular DNA presenting in test samples have been demonstrated to have no impact on SV40 quantification. This method offers significant advantages over other PCR methods and cell-based infectivity assays currently available for SV40 detection and quantification. The availability of this method should greatly facilitate the pathogenic investigation of SV40, as well as viral clearance evaluations required for the development of new biological products.

DNA, Viral↗
PubMed2000

Mouse homolog of Saccharomyces cerevisiae spo11 is induced in normal mu(+)B-cells by stimuli that cause germline C(H) transcription and subsequent class switch recombination.

The first step of Ig heavy chain class switch recombination (CSR) is considered to be DNA double strand break (DSB) formation in the two switch (S) regions (S(mu) and downstream S(H)), although the underlying mechanism is unknown. Recently, it has been demonstrated that at least Spo11, a homolog of the novel type II topoisomerase (topo VI) that catalyzes DSB formation, is involved in the initiation of meiotic recombination of Saccaromyces cerevisiae. In the present study, we examined whether the mouse homolog of Spo11 is induced in normal mouse mu(+)B-cells by stimuli that cause an early step of CSR, germline C(H) transcription, and subsequent CSR. Two CSR systems were used: IgA CSR induced by all-trans retinoic acid, IL-5, and LPS, and IgG1 CSR induced by IL-4 and LPS. Germline transcript and mouse Spo11 expression were analyzed by RT-PCR. In both systems, first germline transcripts were clearly detected on day 2 and then Spo11 was detected on day 3, increasing thereafter with time. The time course of changes in Spo11 expression coincided with that of CSR. Spo11 seems to be induced by CSR-inducing stimuli, regardless of the direction of CSR. These results suggested that mouse Spo11 might participate in the initiation step of CSR.

Animals↗
PubMed2001

Growing dictyate oocytes, but not early preimplantation embryos, of the mouse display high levels of DNA homologous recombination by single-strand annealing and lack DNA nonhomologous end joining.

We have investigated the ability of growing dictyate oocytes and early preimplantation embryos of the mouse to process extrachromosomal DNA molecules with free ends by intranuclearly microinjecting DNA fragments containing a region of homology of various extent at either the 5' or 3' terminus. Homologous recombination of these fragments by single-strand annealing (SSA), but not other DNA recombination/joining mechanisms, resulted in the formation of a full-length hsp-lacZ-pA fusion gene that was transcriptionally activated by heat shock in growing oocytes and spontaneously at the early two-cell stage in the embryos, making it possible to quantitatively evaluate SSA activities of these cells by the beta-galactosidase produced. SSA activities of oocytes and embryos were similar in their general properties and in the activity levels observed with saturating amounts of DNA. However, embryo SSA was almost one order of magnitude less effective than that of oocytes. Oocyte and embryo 5' --> 3' exonuclease (a key function of the SSA pathway) and DNA nonhomologous end joining (NHEJ) activities were also investigated using an asymmetric PCR assay. Results showed that NHEJ is lacking in oocytes and is very prominent in the embryos, where it competes with SSA for the injected DNA.

Animals↗
PubMed1993

Strand-specific binding to duplex DNA ends by the subunits of the Escherichia coli RecBCD enzyme.

RecBCD enzyme of Escherichia coli unwinds DNA from duplex DNA ends to produce single-stranded DNA, a central intermediate in the major (RecBCD) pathway of homologous recombination. To help elucidate the mechanism of unwinding we studied the complex of RecBCD enzyme bound to duplex DNA ends in the absence of ATP, a cofactor required for unwinding. In this complex the terminal 16 or 17 nucleotides of the 3' terminated strand and the terminal 20 or 21 nucleotides of the 5' terminated strand were protected from DNase I cleavage. u.v.-irradiation of the complex cross-linked the RecB subunit to the 3' terminated strand and the RecC and RecD subunits of the 5' terminated strand. Studies using pyridoxal 5-phosphate, an inhibitor of the RecBCD enzyme, corroborated the cross-linking studies and revealed a conformational change in the enzyme upon binding to DNA. Based on these results and proposals by others, we present a model for the mechanism of DNA unwinding by RecBCD enzyme.

Base Sequence↗
PubMed1993

Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chi-specific, but not non-specific, nuclease activity.

RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced approximately 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at chi sites, which are hotspots for genetic recombination. Since this interaction with chi significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.

Adenosine Triphosphate↗
PubMed1993

The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro.

The Escherichia coli recB2109CD enzyme displays a defect in homologous recombination. In vitro, it possesses significant levels of non-specific nuclease activity but is deficient in chi-dependent nicking activity. To determine whether an alteration in helicase activity contributes further to its in vivo defect, the ability of recB2109CD enzyme to unwind dsDNA was examined. The mutant enzyme is able to unwind DNA but has a kcat which is one-third that of the wild-type enzyme. While the Km for DNA ends of the wild-type and mutant enzymes at low NaCl concentrations are essentially equivalent, the Km for ATP of recB2109CD enzyme is nearly six times greater. The processivity of unwinding (i.e. the average length of DNA unwound before recB2109CD enzyme dissociates from the DNA substrate) at 1 mM-Mg2+ ion and 1 mM-ATP is approximately 13 kb/end, whereas that of wild-type recBCD enzyme is 30 kb/end. In an assay which requires the co-ordinate actions of the recBCD, recA, and SSB proteins, joint molecule formation in the presence of recB2109CD enzyme is up to sixfold slower and proceeds to a lower extent than that mediated by the wild-type enzyme. We conclude that although the reduced helicase activity of the mutant recBCD enzyme may contribute to its recombination deficiency, its defect in the chi-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.

Adenosine Triphosphate↗
PubMed1994

Production of UV-induced frameshift mutations in vitro by DNA polymerases deficient in 3'-->5' exonuclease activity.

In order to study the conversion of UV lesions into frameshift and base substitution mutations, M13mp2 phage DNA was altered by the addition of extra pyrimidines, or by construction of a nonsense codon preceded by a run of pyrimidines within the beta-galactosidase complementing region. The normal sequence 5' GTC GTT TTA CAA 3' was changed to GTC GTT T TTA CAA (MIDT) or GTC GTT C TTA CAA (MIDC) to study frameshifts and to GTC GTT CTT TAA (OCHRE) to study reversion of the ochre (TAA) codon. Escherichia coli pol I Kf and T7 DNA polymerase mutant enzymes devoid of 3'-->5' exonuclease activity produced UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. Removal of cyclobutane dimers with photolyase before in vitro synthesis did not greatly affect mutant frequency although such treatment led to significantly increased DNA synthesis by the wild-type T7 DNA polymerase on UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA produced by the delta 28 T7 DNA polymerase were mainly by base substitution in the TAA codon. About half of the E. coli Kf exo- enzyme ochre revertants had a TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity gave revertant frequencies that correlated better with published values of enzyme velocity than with exonuclease activity or with measured bypass synthesis. Our data indicate that loss of proofreading activity increases the frequency of UV-induced frameshifts, but lack of such activity is not sufficient for their production. We suggest that frameshifts occur more frequently when nucleotide addition opposite the lesion is slow. The same lesion can give rise to a different spectrum of mutations depending on the polymerase.

Bacteriophage M13↗