Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Uracil is incorporated into newly synthesized DNA by mutants of Escherichia coli with reduced levels of dUTPase (dUTP nucleotidohydrolase; EC 3.6.1.23). Excision-repair of the incorporated uracil results in the generation of labeled DNA fragments that appear after brief pulses with [(3)H]thymidine [Tye, B-K., Nyman, P.-D., Lehman, I. R., Hochhauser, S. & Weiss, B. (1977) Proc. Natl. Acad. Sci. USA 74, 154-157]. Uracil is also incorporated into the newly synthesized DNA of strains of E. coli that contain normal levels of dUTPase. DNA fragments generated by the postreplication excision-repair of uracil may therefore contribute to the pool of nascent DNA (Okazaki) fragments that normally appear in wild-type strains. Discontinuous DNA replication has been examined in the absence of uracil excision by comparing Okazaki fragments in strains that are defective in DNA polymerase I (polA(-)) and polA(-) strains that are also defective in uracil N-glycosidase, an enzyme required for the excision-repair of uracil in DNA (polA(-)ung(-)). Little or no difference was detected in the level of Okazaki fragments in the polA(-) strain as compared with the polA(-)ung(-) strain. Thus, the uracil-induced cleavage of DNA cannot be the sole mechanism for the generation of Okazaki fragments. Mutants that are defective both in dUTPase and in uracil N-glycosidase incorporate uracil into their DNA with a high frequency (up to 1 per 100 nucleotides). These uracil residues, once incorporated, persist in the DNA without an adverse affect on the growth of the cells.
 ...
PMID:Uracil incorporation: a source of pulse-labeled DNA fragments in the replication of the Escherichia coli chromosome. 20 31

A number of mutant strains of Escherichia coli have been examined for their sensitivity to nitrous acid and in some instances to methylmethanesulfonate. All ung- mutants tested are abnormally sensitive to nitrous acid. Since the ung mutation is phenotypically expressed as a defect in uracil DNA glycosidase, this observation supports the contention that treatment of cells with nitrous acid causes deamination of cytosine to uracil. In addition the observed sentitivity indicates that the ung gene is involved in the repair of uracil in DNA. Studies with other mutants suggest that both exonuclease III and DNA polymerase I of E. coli are involved in the repair of nitrous acid damage in vivo.
 ...
PMID:Repair of nitrous acid damage to DNA in Escherichia coli. 33 52

We describe an efficient method for the construction of small-insert genomic libraries enriched for highly polymorphic, simple sequence repeats. With this approach, libraries in which 40-50% of the members contain (CA)n repeats are produced, representing an approximately 50-fold enrichment over conventional small-insert genomic DNA libraries. Briefly, a genomic library with an average insert size of less than 500 base pairs was constructed in a phagemid vector. Amplification of this library in a dut ung strain of Escherichia coli allowed the recovery of the library as closed circular single-stranded DNA with uracil frequently incorporated in place of thymine. This DNA was used as a template for second-strand DNA synthesis, primed with (CA)n or (TG)n oligonucleotides, at elevated temperatures by a thermostable DNA polymerase. Transformation of this mixture into wild-type E. coli strains resulted in the recovery of primer-extended products as a consequence of the strong genetic selection against single-stranded uracil-containing DNA molecules. In this manner, a library highly enriched for the targeted microsatellite-containing clones was recovered. This approach is widely applicable and can be used to generate marker-selected libraries bearing any simple sequence repeat from cDNAs, whole genomes, single chromosomes, or more restricted chromosomal regions of interest.
 ...
PMID:Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences. 131 88

The generalized mismatch repair system of Streptococcus pneumoniae (the Hex system) can eliminate base pair mismatches arising in heteroduplex DNA during transformation or by DNA polymerase errors during replication. Mismatch repair is most likely initiated at nicks or gaps. The present work was started to examine the hypothesis that strand discontinuities arising after removal of uracil by uracil DNA-glycosylase (Ung) can be utilised as strand discrimination signals. We show that mismatch repair efficiency is enhanced 3- to 6-fold when using uracil-containing DNA as donor in transformation. In order to assess the contribution of Ung to nascent strand discrimination for postreplication mismatch repair, we developed a positive selection procedure to isolate S. pneumoniae Ung- mutants. We succeeded in isolating Ung- mutants using this procedure based on chromosomal integration of uracil-containing hybrid DNA molecules. Cloning and characterization of the ung gene was achieved. Comparison of spontaneous mutation rates in strains either proficient or deficient in mismatch and/or uracil repair gave no support to the hypothesis that Ung plays a major role in targeting the Hex system to neosynthesized DNA strands. However Ung activity is responsible for the increased efficiency of mismatch repair observed in transformation with uracil-containing DNA. In addition Ung is involved in repair of bisulfite-treated transforming DNA.
 ...
PMID:Uracil-DNA glycosylase affects mismatch repair efficiency in transformation and bisulfite-induced mutagenesis in Streptococcus pneumoniae. 194 30

Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E. coli strains. Uracil-containing DNA was prepared after growth of phage in an E. coli strain dut- ung-. The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase. Ung+ and ung- E. coli cells were transformed by DNA. In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50%. Absolute number of mutants was higher in ung+ cells. The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.
 ...
PMID:[UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells]. 268 22

M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.
 ...
PMID:Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides. 635 60

Plasmids were constructed with guanine (G) or O6-methyl- (m6G), O6-ethyl-(e6G), or O6-benzyl- (b6G) guanine in the initiation codon (ATG) of the lacZ' gene. Four deoxyuridine residues were incorporated near the modified guanine in the complementary strand. The deoxyuridine-containing plasmids exhibited similarly high transformation efficiencies in ung- Escherichia coli, although the frequency of mutations induced by m6G, e6G, and b6G residues was relatively low. Treatment of the plasmids with uracil-DNA glycosylase (UDG), to remove the uracil residues, or UDG and exonuclease III, to create a gap in the deoxyuridine-containing strand, reduced transformation efficiency for adduct-containing plasmids but did not affect transformation efficiency for control plasmids. However, the same treatments dramatically enhanced mutagenesis by m6G, e6G, and b6G. These results were consistent with blockage of replication by the modified guanines in double-stranded plasmids resulting in preferential replication of the complementary strand. Replication past the modified guanines was forced in the gapped plasmids. The frequency of modified guanine-induced mutations in gapped vectors was similar in strains of E. coli that were proficient in DNA polymerase III but deficient in either DNA polymerase I or II or both polymerase I and II suggesting either that polymerase III was primarily responsible for adduct bypass in all strains or that the probability of base misinsertion during bypass by either polymerase I or II was similar to that for polymerase III. Repair studies with gapped plasmids indicated that m6G was subject to repair by Ada methyltransferase and to postreplication processing by methylation-directed mismatch repair. Neither e6G nor b6G were similarly repaired.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Mutagenesis in Escherichia coli by three O6-substituted guanines in double-stranded or gapped plasmids. 761 34

Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediate-early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.
 ...
PMID:Characterization of the uracil-DNA glycosylase activity of Epstein-Barr virus BKRF3 and its role in lytic viral DNA replication. 1710 49

Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO(3)-inducing cytosine deamination than wild-type strain.
 ...
PMID:Cloning and characterization of uracil-DNA glycosylase and the biological consequences of the loss of its function in the nematode Caenorhabditis elegans. 1852 57