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PubMed2005

Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins.

We describe a new method for the assay of sequence-specific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNA footprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNA-binding proteins. We named this method as exonuclease III (ExoIII) protection assay with FRET probe. The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion, resulting in high FRET. However, if the target protein does not exist, the FRET pair on the naked FRET probe will be degraded by ExoIII, resulting in low FRET. Three kinds of recombinant transcription factors including NF-kappaB, SP1 and p50, and the target protein of NF-kappaB in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.

Cell Nucleus↗
PubMed2004

High-throughput SNP genotyping by combining exonuclease III, nuclease S1, and acridine-bearing PNA.

Recently we reported a precise and straightforward method for genotyping of single nucleotide polymorphisms (SNPs) in double-stranded DNA substrates. The substrates were consecutively treated by two enzymes (exonuclease III and nuclease S1) in the presence of peptide nucleic acid (PNA) probes, and the resultant DNA fragments were analyzed by mass spectroscopy. Here, the selectivity of DNA scission by nuclease S1 has been greatly improved by attaching an acridine to the PNA probes. Modification at the C-terminus was especially effective. The number of DNA fragments formed has been greatly decreased so that double-stranded DNA substrates can be genotyped by using the two enzymes still more easily and precisely.

Acridines↗
PubMed2025

A bacterial PrimPol-reverse transcriptase hybrid protein has a proofreading exonuclease activity that can be transferred to other reverse transcriptases.

Gene disruption analysis revealed that an E. coli PPRT protein, which has an N-terminal Primase-Polymerase (PrimPol) domain fused to a group II intron-like reverse transcriptase (RT) domain followed by a long C-terminal domain (CTD), contributes to a cellular oxidative DNA damage response in addition to its previously described function in phage defense. Biochemical analysis showed that the PrimPol domain has an error-prone DNA polymerase activity that enables read through of oxidation-induced DNA damage. Surprisingly, we found that the RT-like domain, in addition to synthesizing protein-primed DNAs for phage defense, has a 3' to 5' DNA exonuclease activity that functions in proofreading DNAs synthesized by the PrimPol domain. Extending these findings, we identified structural features that contribute to this proofreading activity, enabling us to associate it with both a group II intron-encoded and retroviral RT and suggesting general methods for incorporating proofreading activity into RTs.

DNA sequencing↗
PubMed1996

The 3' end of yeast 5.8S rRNA is generated by an exonuclease processing mechanism.

Eukaryotic rRNAs (with the exception of 5S rRNA) are synthesized from a contiguous pre-rRNA precursor by a complex series of processing reactions. Final maturation of yeast 5.8S rRNA involves processing of a 3'-extended, 7S precursor that contains approximately 140 nucleotides of the internal transcribed spacer 2 (ITS2) region. In yeast strains carrying the temperature-sensitive (ts) rrp4-1 mutation, 5.8S rRNA species were observed with 3' extensions of variable length extending up to the 3' end of the 7S pre-rRNA. These 3'-extended 5.8S rRNA species were observed at low levels in rrp4-1 strains under conditions permissive for growth and increased in abundance upon transfer to the nonpermissive temperature. The RRP4 gene was cloned by complementation of the ts growth phenotype of rrp4-1 strains. RRP4 encodes an essential protein of 39-kD predicted molecular mass. Immunoprecipitated Rrp4p exhibited a 3'-->5' exoribonuclease activity in vitro that required RNA with a 3'-terminal hydroxyl group and released nucleoside 5' monophosphates. We conclude that the 7S pre-rRNA is processed to 5.8S rRNA by a 3'-->5' exonuclease activity involving Rrp4p. Homologs of Rrp4p are found in both humans and fission yeast Schizosaccharomyces pombe (43% and 52% identity, respectively), suggesting that the mechanism of 5.8S rRNA 3' end formation has been conserved throughout eukaryotes.

Amino Acid Sequence↗
PubMed1989

A 3' exonuclease activity degrades the pseudogene 5S RNA transcript and processes the major oocyte 5S RNA transcript in Xenopus oocytes.

Transcription of the major oocyte 5S RNA gene (o) and pseudogene (psi) of Xenopus laevis yields different RNAs with three different homologous systems: oocyte microinjection, whole oocyte extract, and fractionated TFIIIA + TFIIIB + TFIIIC components. Those peculiar results are caused by a 3' RNA exonuclease activity, which is inhibited in the oocyte extract, that rapidly degrades the pseudogene 5S RNA but does not degrade as readily the chimeric RNA transcripts generated by HindIII-truncated 5S RNA pseudogenes. The same, or a similar, RNase activity processes the 130- and the 142-base-long transcripts of the major oocyte 5S RNA gene into mature 120-base-long 5S RNA. We performed site-specific mutagenesis on the somatic 5S RNA gene and changed specific nucleotides on the somatic 5S RNA. These studies indicated that the structure that confers stability to the 5S RNA in vivo and in vitro is the 9-bp helix formed in 5S RNA, but not in psi 5S RNA, by the complementary 5' and 3' ends of the molecule.

Animals↗
PubMed2003

Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder.

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.

Bacteriophage lambda↗
PubMed1997

Toroidal structure of lambda-exonuclease.

Structure determination at 2.4 angstrom resolution shows that lambda-exonuclease consists of three subunits that form a toroid. The central channel is funnel shaped, tapering from an inner diameter of about 30 angstroms at the wider end to 15 angstroms at the narrow end. This is adequate to accommodate the DNA substrate and thus provides a structural basis for the ability of the enzyme to sequentially hydrolyze thousands of nucleotides in a highly processive manner. The results also suggest the locations of the active sites and the constraints that limit cleavage to a single strand.

Bacteriophage lambda↗
PubMed1995

Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA.

Simple repetitive DNA sequences are unstable in human colorectal cancers and a variety of other cancers. Mutations in the DNA mismatch repair genes MSH2, MLH1, and PMS1 result in elevated rates of spontaneous mutation and cause a marked increase in the instability of simple repeats. Compared with the wild type, a null mutation in the yeast RTH1 gene, which encodes a 5' to 3' exonuclease, was shown to increase the rate of instability of simple repetitive DNA by as much as 280 times and to increase the spontaneous mutation rate by 30 times. Epistasis analyses were consistent with the hypothesis that this RTH1-encoded nuclease has a role in the MSH2-MLH-1-PMS1 mismatch repair pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Real-time PCR detection of Brucella abortus: a comparative study of SYBR green I, 5'-exonuclease, and hybridization probe assays.

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches-SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)-were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.

Benzothiazoles↗
PubMed1985

Spontaneous induction of colicin E1 in Escherichia coli strains deficient in both exonucleases I and V.

Colicin E1 synthesis is spontaneously induced in pRSF2124-carrying strains of Escherichia coli deficient in exonucleases I (sbcB) and V (recB recC). In contrast, the specific activity of beta-lactamase, which is also encoded by pRSF2124, is not affected by the absence of these enzymes. These results suggest that colicin E1 induction is specific and does not result either from a significant change in overall plasmid transcription or copy number. Furthermore, the level of spontaneous induction was similar to that obtained with mitomycin C.

Colicins↗
PubMed1986

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3'----5' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).

Base Sequence↗
PubMed1989

Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase.

The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.

Apurinic Acid↗
PubMed2002

Escherichia coli DNA polymerase III can replicate efficiently past a T-T cis-syn cyclobutane dimer if DNA polymerase V and the 3' to 5' exonuclease proofreading function encoded by dnaQ are inactivated.

Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coli in the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (DeltaumuDC) strains if the 3' to 5' exonuclease proofreading activity of the Pol III epsilon subunit was also disabled by mutD5. This observation might be explained in at least two ways. In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases. Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function. We have investigated this issue by examining dimer bypass frequencies in DeltaumuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations. Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V. Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself. This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally.

DNA Polymerase III↗
PubMed2002

Bacillus subtilis YhaM, a member of a new family of 3'-to-5' exonucleases in gram-positive bacteria.

A strain of Bacillus subtilis lacking two 3'-to-5' exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase R, was used to purify another 3'-to-5' exoribonuclease, which is encoded by the yhaM gene. YhaM was active in the presence of Mn(2+) (or Co(2+)), was inactive in the presence of Mg(2+), and could also degrade single-stranded DNA. The half-life of bulk mRNA in a mutant lacking PNPase, RNase R, and YhaM was not significantly different from that of the wild type, suggesting the existence of additional activities that can participate in mRNA turnover. Sequence homologues of YhaM were found only in gram-positive organisms. The Staphylococcus aureus homologue, CBF1, which had been characterized as a double-stranded DNA binding protein involved in plasmid replication, was also shown to be an Mn(2+)-dependent exoribonuclease. YhaM protein has a C-terminal "HD domain," found in metal-dependent phosphohydrolases. By structure modeling, it was shown that YhaM also contains an N-terminal "OB-fold," present in many oligosaccharide- and oligonucleotide-binding proteins. The combination of these two domains is unique. Thus, YhaM and 10 related proteins from gram-positive organisms constitute a new exonuclease family.

Amino Acid Sequence↗
PubMed1999

Rapid identification of up to three Candida species in a single reaction tube by a 5' exonuclease assay using fluorescent DNA probes.

We used fungus-specific PCR primers and species-specific DNA probes to detect up to three Candida species in a single reaction tube by exploiting the 5' to 3' exonuclease activity of Taq DNA polymerase. Probes to the internal transcribed spacer region of the rRNA gene were labeled at the 5' end with one of three fluorescent reporter dyes, 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxy-fluorescein (TET), or hexachloro-6-carboxy-fluorescein (HEX), and at the 3' end with a quencher dye, 6-carboxy-tetramethyl-rhodamine. During PCR amplification, each reporter dye emits a characteristic wavelength as it is cleaved from its specific target DNA and from the quencher dye. Therefore, signals from up to three probes can be detected simultaneously during the PCR assay. Six probes were designed for use in this study: CA-FAM, CT-TET, and CP-HEX were added to one tube to simultaneously detect the typically fluconazole-sensitive species C. albicans, C. tropicalis, and C. parapsilosis, respectively. CG-FAM and CK-TET were added to a second tube to simultaneously detect the typically more innately fluconazole-resistant species C. glabrata and C. krusei, respectively. All-CAN-TET, a Candida genus probe, was added to a third tube to detect DNAs from all Candida species tested. DNAs recovered from 61 blood culture bottles, including 23 positive for C. albicans, 18 positive for C. glabrata, 6 positive for C. tropicalis, 6 positive for C. krusei, 5 positive for C. parapsilosis, and 3 positive for mixed fungemias, were tested. Control samples included those from blood culture bottles with no growth (n = 10) or from patients with confirmed bacteremia (n = 10). Probes detected and correctly identified the organisms in 58 of 61 specimens (95.1%) and gave no false-positive results. This method is simple and rapid and does not require post-PCR hybridization and incubation steps. It is sensitive and specific for the detection and identification of Candida species from blood culture bottles, including those containing mixtures of Candida species, and should facilitate an earlier specific diagnosis, leading to more appropriately targeted antifungal drug therapy.

Candida↗
PubMed1999

Detection and quantitation of human papillomavirus by using the fluorescent 5' exonuclease assay.

A method for the detection and quantitation of oncogenic human papillomavirus (HPV) was developed by using the fluorescent 5' exonuclease assay. The method is based on the amplification of a 180-bp fragment from the 3' part of the E1 open reading frame in a single PCR with type-specific probes for HPV types 16, 18, 31, 33, and 35. The probes can be used separately or in combinations of up to three probes per assay. Quantitation over a range of 10(1) to 10(6) initial HPV copies was possible by using real-time detection of the accumulation of fluorescence with cycle number. Reconstitution experiments, performed to mimic mixed infections, showed that individual HPV types can be detected down to a ratio of about 1% in a mixture. The performance of the assay depends on DNA quality, the presence of PCR inhibitors, and the number of different probes used simultaneously. This homogeneous assay provides a fast and sensitive way of screening for oncogenic HPV types in biopsy specimens as well as cervical smear samples. The closed-tube nature of the assay and the inclusion of uracil N'-glycosylase reduces cross contamination of PCR products to a minimum. A similar assay for beta-actin was used in parallel for quantitation of genomic DNA. After normalizing the samples for genomic DNA content, the mean number of HPV copies per cell could be calculated.

Cervix Uteri↗
PubMed2002

Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis complex in clinical samples by 5'-exonuclease fluorogenic PCR.

Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5'-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.

DNA, Bacterial↗
PubMed1982

Bacteriophage T7 defective in the gene 6 exonuclease promotes site-specific cleavages of T7 DNA in vivo and in vitro.

Site-specific cleavages of intracellular DNA were demonstrated in bacteriophage T7 6am-infected cells. The sites of the cleavages were located at 46.8 and 68.7% (1% of the T7 DNA length = 400 base pairs) from the left end of the T7 genome. These cleavages required the products of genes 3 (endonuclease), 4 (DNA primase), and 5 (DNA polymerase). However, the product of gene 6 (exonuclease) must be absent. Site-specific cleavage was also shown to occur in vitro in extracts of T7 6am-infected cells, although at a different site: 82.8% from the left end of the T7 genome.

DNA Primase↗