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Biomedical subjects

A F Taylor

Publications and source records attributed to A F Taylor.

47 records · Page 3Linked to original sources
PubMed1985

Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli.

The RecBC enzyme of Escherichia coli promotes genetic recombination of phage or bacterial chromosomes. The purified enzyme travels through duplex DNA, unwinding and rewinding the DNA with the transient production of potentially recombinogenic single-stranded DNA. The studies reported here are aimed at understanding which chromosomal forms allow the entry of RecBC enzyme and hence may undergo RecBC enzyme-mediated recombination. Circular duplex molecules, whether covalently closed, nicked or containing single-stranded gaps of 10 to 774 nucleotides, are not detectably unwound by RecBC enzyme. Linear duplex molecules are readily unwound if they have a nearly flush-ended terminus whose 5' and 3' ends are offset by no more than about 25 nucleotides; molecules with longer single-stranded tails are poorly bound by RecBC enzyme and are infrequently unwound. The single-strand endonuclease activity of RecBC enzyme can slowly cleave gapped circles to produce molecules presumably capable of being unwound. These results provide an enzymatic basis for the recombinogenicity of double-stranded DNA ends established from genetic studies of RecBC enzyme and Chi sites, recognition sites for RecBC enzyme-mediated DNA strand cleavage.

Autoradiography↗
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↗
PubMed1984

Unusual alleles of recB and recC stimulate excision of inverted repeat transposons Tn10 and Tn5.

Precise and nearly precise excision of transposon Tn10 occur by host-mediated processes unrelated to transposition. Both types of excision involve interactions between short (9 or 24 base-pair) direct repeat sequences at or near the termini of the transposon and are stimulated by the large (1,329-base-pair) inverted repeats that form the ends of Tn10. We describe here three mutations of Escherichia coli K-12, designated texA, that enhance excision of Tn10 and of the structurally analogous transposon Tn5. Genetic mapping and complementation analysis show that these mutations are unusual alleles of the recB and recC genes that alter but do not abolish RecBC function. As Tn10 excision normally does not depend on RecA or RecBC functions, texA mutations appear to provide another pathway for excision that depends on altered RecBC function; for one texA allele, excision has become dependent on RecA function as well. The available evidence suggests that texA mutations alter the stimulatory interaction between the inverted repeats of Tn10.

Alleles↗
PubMed1984

Vitamin D metabolite levels in normal children.

Vitamin D metabolite levels were measured in 174 normal children throughout the year. 25-hydroxyvitamin D3 (25(OH)D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) levels showed a seasonal variation; both levels were higher in summer than in winter (p less than 0.001 for both). There was a fall in the 1,25-dihydroxyvitamin D (1,25(OH)2D) level in August and September (p less than 0.001), which coincided with a rise in mean 25(OH)D3 and 24,25(OH)2D3 levels. An inverse correlation was seen between 1,25(OH)2D and 24,25(OH)2D3 levels (r = -0.233; p less than 0.01). 25(OH)D3 levels increased with age only for winter values (less than 3 years, 11.70 +/- 3.98 ng/ml; 3-11 years, 18.38 +/- 1.65 ng/ml; greater than 11 years, 23.60 +/- 4.60 ng/ml) while 1,25(OH)2D and 24,25(OH)2D3 levels did not show an age-related difference. Intake of multivitamins had an interesting effect on 25(OH)D3 and 24,25(OH)2D3 levels in the winter but not in the summer; the endogenous metabolite levels were lower in the vitamin supplemented children [25(OH)D3: 23.05 +/- 7.35 versus 15.77 +/- 5.51 ng/ml, p less than 0.001; 24,25(OH)2D3: 2.30 +/- 1.11 versus 1.66 +/- 0.88 ng/ml, p less than 0.05]. Children studied in the winter who were not receiving supplemental vitamins were older than those who did receive the vitamins (7.26 +/- 2.64 versus 5.42 +/- 3.17 years; p less than 0.01). Sixteen of the children had both winter and summer measurements. Their 25(OH)D3 and 24,25(OH)2D3 levels showed the same seasonal variation as the overall group data, while their 1,25(OH)2D levels showed no consistent pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

24,25-Dihydroxyvitamin D 3↗
PubMed1983

Escherichia coli RecBC pseudorevertants lacking chi recombinational hotspot activity.

Pseudorevertants of an Escherichia coli exonuclease V (RecBC enzyme)-negative mutant have been isolated after ethyl methane sulfonate mutagenesis of a recC73 (presumed missense) mutant. The remedial mutations in each of the four pseudorevertants studied in detail map and complement as recC mutations. By several criteria, such as recombination proficiency, support of phage growth, RecBC nuclease activity, and cell viability, the pseudorevertants appear to have regained partially or completely various aspects of RecBC activity. However, chi recombinational hotspots, which stimulate exclusively the RecBC pathway of recombination, have no detectable activity in lambda vegetative crosses in the pseudorevertants. The properties of these mutants, in which the RecBC pathway of recombination is active yet in which chi is not active, are consistent with the hypothesis that wild-type RecBC enzyme directly interacts with chi sites; alternatively, the mutants may block or bypass the productive interaction of another recombinational enzyme with chi.

Coliphages↗
PubMed1982

Role of exonuclease III in the base excision repair of uracil-containing DNA.

Mutants of Escherichia coli K-12 deficient in both exonuclease III (the product of the xth gene) and deoxyuridine triphosphatase (the dut gene product) are inviable at high temperatures and undergo filamentation when grown at such temperatures. In dut mutants, the dUTP pool is known to be greatly enhanced, resulting in an increased substitution of uracil for thymine in DNA during replication. The subsequent removal of uracil from the DNA by uracil-DNA glycosylase produces apyrimidinic sites, at which exonuclease III is known to have an endonucleolytic activity. The lethality of dut xth mutants, therefore, indicates that exonuclease III is important for this base-excision pathway and suggests that unrepaired apyrimidinic sites are lethal. Two confirmatory findings were as follows. (i) dut xth mutants were viable if they also had a mutation in the uracil-DNA glycosylase (ung) gene; such mutants should not remove uracil from DNA and should not, therefore, generate apyrimidinic sites. (ii) In the majority of the temperature-resistant revertants isolated, viability had been restored by a mutation in the dCTP deaminase (dcd) gene; such mutations should decrease dUTP production and hence uracil misincorporation. The results indicate that, in dut mutants, exonuclease III is essential for the repair of uracil-containing DNA and of apyrimidinic sites.

DNA Repair↗
PubMed1980

Cloning of the dut (deoxyuridine triphosphatase) gene of Escherichia coli.

Through the molecular cloning of DNA, cells were obtained that could produce a 300-fold increased level of deoxyuridine triphosphatase (dUTPase). First, lambda pyrE-dut phages were constructed from restriction endonuclease fragments. They contained a segment of Escherichia coli DNA that spanned the structural genes for dUTPase (dut) and orotidylate pyrophosphorylase (pyrE). The initial isolates demonstrated poor enzyme production and impaired growth. Improved enzyme yields were then obtained from large-plaque derivatives and from mutants with partial deletions of the cloned DNA. The deletion mutants were isolated after the induction of a recombinant prophage whose DNA was too large to be packaged. Finally, a 3.3-kb segment of DNA, containing the dut gene, was transferred to plasmid vectors. The recombinants and their levels of dUTPase overproduction (relative to that of wild type cells) were as follows: a thermoinducible lambda pyrE-dut phage, 45-fold (10-fold for orotidylate pyrophosphorylase); a dut-ColE1 type plasmid, 15-fold; and a thermoinducible dut-lambda-ColE1 chimera, 14-fold before induction and 300-fold after induction.

Bacteriophage lambda↗