scholarly journals Temperature-sensitive mutations in the yeast DNA polymerase I gene.

1987 ◽  
Vol 84 (9) ◽  
pp. 2838-2842 ◽  
Author(s):  
M. Budd ◽  
J. L. Campbell
Keyword(s):  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Dna Polymerase I ◽  
Polymerase I ◽  
I Gene

scholarly journals Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 213-220 ◽  
Author(s):  
L J Reha-Krantz
Keyword(s):  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Rnase H ◽  
Ribonuclease H ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Dna Polymerase I ◽  
E Coli ◽  
Intragenic Complementation ◽  
Polymerase I

Abstract Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.

scholarly journals The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli.

Genetics ◽  
1995 ◽  
Vol 139 (4) ◽  
pp. 1483-1494 ◽  
Author(s):  
Y Cao ◽  
T Kogoma
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Reca Protein ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase I ◽  
Recombination Repair ◽  
Polymerase I

Abstract The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.

scholarly journals Interaction between the plasmid R6K andEscherichia coliwith defective DNA polymerase I

1978 ◽  
Vol 32 (1) ◽  
pp. 25-35 ◽  
Author(s):  
D. J. Tweats ◽  
J. T. Smith
Keyword(s):  
Cell Division ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase I ◽  
E Coli ◽  
R Factor ◽  
Polymerase I

SUMMARYInitial experiments demonstrated that the plasmid R6K cannot be transferred to or maintained readily in theE. coliDNA polymerase I deficient strain JG138polA1. Results withE. coliMM386polA12(R6K), which has a temperature sensitive polymerase I enzyme, showed cell division becomes abnormal when the polymerase I enzyme of the host bacteria is inactivated at the restrictive temperature. Under conditions of polymerase I deficiency, R6K replication, as measured by monitoring R-factor-mediated β-lactamase activity, also becomes abnormal with the loss of multiple R6K copies per cell and the apparent maintenance of a single R-factor copy per cell.

scholarly journals DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 365-376
Author(s):  
M E Budd ◽  
K D Wittrup ◽  
J E Bailey ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Dna Polymerase I ◽  
X Ray ◽  
Temperature Sensitive Mutants ◽  
Polymerase I ◽  
Dna Polymerase Ii

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

scholarly journals Molecular Cloning and Chartacterization DNA Polymerase I from thermophilic Geobacillus SBS 4S strain

2019 ◽  
Author(s):  
farheen aslam ◽  
Saima Iftikhar Bajwa
Keyword(s):  
Recombinant Protein ◽  
Dna Polymerase ◽  
Soluble Fraction ◽  
Dna Polymerase I ◽  
Ammonium Sulphate Precipitation ◽  
Cation Exchange Column ◽  
Geobacillus Kaustophilus ◽  
Cloned Gene ◽  
Polymerase I ◽  
I Gene

Abstract Thermostable DNA polymerases are extensively used in biotechnology and life science applications. DNA Polymerase I was isolated from a hyperthermophile bacteria Geobacillus SBS 4S. Primers were designed using the template sequence of DNA Polymerase I gene of Geobacillus kaustophilus HTA26 strain. Nco 1 and Hind III sites were introduced on the forward and reverse primers respectively. Polymerase I gene of 2.6 Kb was cloned in pTZ57/ RT vector. Cloned gene of Polymerase I was restricted with Nco 1 and Bam H1, and ligated to pET 22b vector. Nco 1 site was used to insert twenty two N-terminal aminoacids (pelB) leader sequence at the start of the gene, which lead the recombinant protein in the periplasmic space, which increases the half life of recombinant protein. pelB fused with DNA Polymerase I produces soluble protein, which was detected after sonication. Sequencing shows that DNA polymerase I consists of 2499 bp with encodes for 832 amino acids, showed 99 % similarity with Geobacillus Kaustophillus . The expression of pelB fused DNA Polymerase I was optimized at different concentrations of IPTG and lactose. Highest expression was observed with 0.5mM IPTG and 20mM lactose. After Harvesting and sonication of BL21 codon plus cells, Polymerase I was produced in the soluble fraction. The supernatant containing the protein of interest, was separated after centrifugation at 10,000 rpm for 20 min. The protein was purified by ammonium sulphate precipitation and cation exchange column. The activity of purified DNA Polymerase I was checked by PCR reaction.

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