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At least 145 records · Page 8Linked to original sources
PubMed1992

Involvement of RecB-mediated (but not RecF-mediated) repair of DNA double-strand breaks in the gamma-radiation production of long deletions in Escherichia coli.

Experiments were designed to determine the association between the repair of gamma-radiation-induced DNA double-strand breaks (DSB) and the induction of 700-1000 bp long deletions (Lac(-)----Lac+), base substitutions (leuB19----Leu+), and frameshifts (trpE9777----Trp+) in Escherichia coli K-12. Over the range of 2.5-20 krad, deletions were induced with linear kinetics, as has been shown for the induction of DSB, while the induction kinetics of base substitutions and frameshifts were curvilinear. Like the repair of DSB, deletion induction showed an absolute requirement for an intact recB gene as well as a dependency on the type of preirradiation growth medium; these requirements were not seen for base substitutions or frameshifts. In addition, about 80% of the spontaneous deletions were absent in the recB21 strain. A recC1001 mutation, which confers a 'hyper-Rec' phenotype, increased the rate of gamma-radiation-induced deletions as well as the low-dose production of base substitutions and frameshifts. A recF143 mutation increased the yield of gamma-radiation-induced deletions without increasing base substitutions or frameshifts. A mutS mutation markedly enhanced the gamma-radiation induction of frameshifts, and had a slight effect on base substitutions, but did not affect the induction of deletions. Resistance to gamma-irradiation and the capacity to repair DSB (albeit at about half the normal rate) were restored to the radiosensitive recB21 strain by the addition of the sbcB21 and sbcC201 mutations. However, the radioresistant recB sbcBC strain, which is recombination proficient via the RecF pathway, was still grossly deficient in the ability to produce deletions. A model for deletion induction as a by-product of the recB-dependent (Chi-dependent) repair of gamma-radiation-induced DSB is discussed, as is the inability to detect deletions in cells that use only the recF-dependent (Chi-independent) mechanism to repair DSB.

Bacterial Proteins↗
PubMed1996

'External' proofreading of DNA replication errors and mammalian autonomous 3'-->5'exonucleases.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. The base substitutions misinserted by these polymerases could be eliminated with two kinds of an 'external' proofreading carried out (1) by the 3'-->5' exonuclease function intrinsic to DNA polymerases delta and epsilon or/and (2) by the autonomous 3'-->5' exonucleases non-associated covalently with DNA polymerases. DNA polymerases delta and epsilon can be separated from autonomous 3'-->5' exonucleases by means of sedimentation. Ultracentrifugation of the nuclear extracts and cytosols from normal and regenerating rat liver as well as from total embryos has shown the bulk of the cellular 3'-->5' exonucleolytic activity is due to autonomous nucleases. Moreover, the level of such a specific activity correlates with the replicative status of the organs from adult animals: spleen > regenerating liver > normal liver > cardiac muscle > brain, maximum difference being an order of magnitude. In addition, autonomous exonucleases were shown to be the constituents of the multienzyme forms of DNA polymerases alpha and beta. Hence, autonomous 3'-->5' exonucleases seem to be the principal participants in an 'external' proofreading.

Animals↗
PubMed1983

Characterization of human cytomegalovirus-induced DNA polymerase and the associated 3'-to-5', exonuclease.

A DNA polymerase activity induced by human cytomegalovirus (HCMV) was separated from host cell DNA polymerase and purified by phosphocellulose and DNA-cellulose column chromatography. The DNA polymerase activity was strongly inhibited by phosphonoacetic acid, aphidicolin, araATP, and N-ethylmaleimide, but it was resistant to 2',3'-dideoxyTTP. The sensitivity of HCMV-induced DNA polymerase to these reagents resembles that of host cell DNA polymerase alpha. However, HCMV-induced DNA polymerase activity was stimulated several fold by 100 mM ammonium sulfate, by which DNA polymerase alpha activity was strongly inhibited. Furthermore, it was found that a 3'-to-5' exonuclease activity was tightly associated with the HCMV-induced DNA polymerase. The exonuclease was also stimulated by ammonium sulfate, was inhibited by phosphoacetic acid, and it preferred single-stranded DNA as a substrate. The results suggest that the 3'-to-5' exonuclease may play a role in proofreading in the polymerization process as an integral part of the HCMV-induced DNA polymerase.

Ammonium Sulfate↗
PubMed1986

Effect of aphidicolin and nucleotide analogs on the phage phi 29 DNA polymerase.

The drugs aphidicolin and the nucleotide analogs butylanilino dATP, butylphenyl dGTP, and butylphenyl rGTP inhibited the protein-primed replication of phi 29 DNA-protein p3 in the presence of purified terminal protein p3 and phi 29 DNA polymerase p2. The effect of aphidicolin was mainly on the polymerization reaction by decreasing the rate of elongation. The nucleotide analogs inhibited both the formation of the p3-dAMP initiation complex and its further elongation, the latter being also due to a decrease in the elongation rate. When assayed with the phi 29 DNA polymerase as the only protein, all the drugs inhibited polymerization on activated DNA as well as the 3'----5' exonuclease activity of the polymerase, indicating that the target of the drugs is the phi 29 DNA polymerase itself.

Adenosine Triphosphate↗
PubMed1987

Sequence of the bacteriophage P22 anti-recBCD (abc) genes and properties of P22 abc region deletion mutants.

The nucleotide sequence of a segment of the bacteriophage P22 chromosome to the left (downstream in the PL operon) of the erf gene was determined. Previous studies (A. C. Fenton and A. R. Poteete, 1984, Virology 134, 148-160) have shown that this region encodes a function that is required for efficient growth of P22 in wild-type, but not in recB- Salmonella. The gene or genes encoding this function were designated abc (anti-recBCD). The DNA sequence reveals three open reading frames that potentially encode polypeptides with molecular weights of 10,900, 11,600, and 6600 (in order of transcription). P22 deletion mutants lacking each of the open reading frames were constructed. In addition, plasmids were constructed placing each of the open reading frames under control of the lac UV5 promoter. The phenotypes of the deletion mutants, and the results of plasmid-phage complementation tests, indicate that Abc activity depends primarily on sequences that encode the 11.6-kDa polypeptide; the 10.9-kDa polypeptide-encoding sequence makes a minor contribution to Abc activity as well. These sequences have been designated abc2 and abc1, respectively. The 6.6-kDa polypeptide is apparently uninvolved.

Amino Acid Sequence↗
PubMed1986

The enzymatic basis of cyclophosphamide specificity.

Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP. Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP). Protein-S-CP stores activated CP in a highly stable form. From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days. From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells. On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity. No spontaneous toxicogenation of activated CP was noted under in vivo conditions. 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products. Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo. 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E. coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate. In experiments with DNA polymerase I from E. coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme. This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase. Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated. Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target. In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.

Animals↗
PubMed1993

In situ detection of DNA-metabolizing enzymes following polyacrylamide gel electrophoresis.

We have presented several protocols for producing an in situ activity gel that allows detection of various DNA-metabolizing enzymes. Both nondenaturing polyacrylamide and SDS-polyacrylamide activity gel electrophoresis procedures were detailed. Combining the use of defined [32P]DNA substrates with product analysis, these procedures detected a wide spectrum of enzymatic activities. The ability to detect 7 different catalytic activities of 15 different enzymes provides encouragement for expanded applications. It is hoped that others will find this technique applicable for detecting these enzymes and other activities in different biological systems. The modification of DNA in situ and the creation of intermediate substrates within activity gels should prove extremely useful for dissecting the enzymatic steps of DNA replication, repair, recombination, and restriction, as well as the metabolic pathways of other nucleic acids.

Bacteriophage M13↗
PubMed1985

Chi-dependent DNA strand cleavage by RecBC enzyme.

Chi sites enhance in their vicinity homologous recombination by the E. coli RecBC pathway. We report here that RecBC enzyme catalyzes Chi-dependent cleavage of one DNA strand, that containing the Chi sequence 5'G-C-T-G-G-T-G-G3'. Chi-specific cleavage is greatly reduced by single base pair changes within the Chi sequence and by mutations within the E. coli recC gene, coding for a RecBC enzyme subunit. Although cleavage occurs preferentially with double-stranded DNA, the product of the reaction is single-stranded DNA. These results demonstrate the direct interaction of RecBC enzyme with Chi sites that was inferred from the genetic properties of Chi and recBC, and they support models of recombination in which Chi acts before the initiation of strand exchange.

Base Sequence↗
PubMed1985

RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting.

Homologous recombination by the E. coli RecBC pathway occurs at elevated frequency near Chi sites. We reported previously that Chi induces RecBC enzyme to cleave one DNA strand--that containing the Chi sequence 5'G-C-T-G-G-T-G-G3'. We report here that the Chi-dependent cleavage occurs four, five, or six nucleotides to the 3' side of the Chi octamer and produces nicks with 3'-OH and 5'-PO4 groups. Chi-dependent cleavage occurs if RecBC enzyme approaches the Chi sequence from the right, but not if it approaches only from the left, during unwinding of the duplex DNA substrate. A single RecBC enzyme molecule appears to cleave the DNA and to release part of it as a single-stranded fragment. These and previous results indicate that Chi-dependent cleavage is concomitant with DNA unwinding by RecBC enzyme and provide an enzymatic basis for the orientation-dependence of Chi recombinational hotspot activity. These observations demonstrate a key step of a proposed model of recombination in which RecBC enzyme produces a potentially invasive single-stranded DNA tail extending from Chi to its left. We discuss the relation between the action of Chi sites and that of special sites enhancing eukaryotic recombination.

Base Sequence↗
PubMed1991

Homologous pairing in vitro stimulated by the recombination hotspot, Chi.

Genetic recombination in Escherichia coli is stimulated at DNA sequences known as Chi sites, 5'-GCT-GGTGG-3'. We describe the in vitro formation of homologously paired joint molecules that is dependent upon this recombination hotspot. Chi-dependent joint molecule formation requires RecA, RecBCD, and SSB proteins and a Chi site in the donor linear dsDNA. The donor dsDNA is unwound by RecBCD enzyme, and the invasive strand is generated by nicking at Chi. This Chi-dependent invading strand must contain homology to the recipient supercoiled DNA substrate at its newly formed 3' end for efficient joint molecule formation. Action at Chi generates invasive ssDNA from the 5' but not the 3' side of Chi, suggesting that the nuclease activity of RecBCD enzyme is attenuated upon encountering a Chi site. These results support the view that RecBCD enzyme action can precede RecA protein action and reconcile the seemingly opposing degradative and recombination functions of RecBCD enzyme.

Base Sequence↗
PubMed1993

The recombination hotspot chi is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme.

The RecBCD enzyme is a multifunctional enzyme that is essential for homologous recombination in E. coli. In vitro, the RecBCD enzyme degrades linear double-stranded DNA nonspecifically during the process of unwinding the double-stranded DNA. Here we demonstrate that this DNA degradation is asymmetric, with the strand that is 3' terminal at the entry site of RecBCD enzyme being degraded much more vigorously than the 5' terminal strand. Furthermore, interaction with the recombination hotspot chi causes an attenuation of the nuclease activity but not of the helicase activity and is accompanied by a pause of RecBCD enzyme at the chi site. These results demonstrate that chi is a unique regulatory element that acts by controlling the degradative function of RecBCD enzyme and, thereby, enhancing its recombination function.

DNA Helicases↗
PubMed1994

DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions.

Homologous recombination-dependent DNA replication (RDR) of a lambda cos site-carrying plasmid is demonstrated in E. coli cells when the cells express lambda terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot chi. Chi is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the theta mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (oriMs) on the chromosome.

Bacterial Proteins↗