Bacillus subtilis bacteriophase PBS2-induced DNA polymerase: its purification and assay characteristics

Journal Article · Sun Dec 10 04:00:00 EST 1978 · J. Biol. Chem.; (United States)

OSTI ID:6803743

Hitzeman, R A; Price, A R

The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 (whose DNA contains uracil instead of thymine) has been purified and characterized for its specificity. The enzyme requires a high ionic strength for optimal stability and activity and is sensitive to various anions and to sulfhydryl reagents. Both dUTP and dTTP are incorporated efficiently as substrates and are competitive inhibitors at the same active site. The apparent K/sub m/ and K/sub i/ values are about 6 ..mu..m for dTTP and 15 ..mu..m for dUTP, when denatured, uracil-containing PBS2 DNA is used as template. The apparent K/sub m/ values are lower with denatured, thymine-containing B, subtilis or salmon sperm DNA (3.9 ..mu..m for dUTP and 2.6 ..mu..m for dTTP). The PBS2 enzyme works best on denatured DNA, on double-stranded DNA activated by DNase to produce gaps, or on primed homopolymeric DNA. Using denatured DNA preparations of average molecular weight 6.2 million, the apparent K/sub m/ values are 270 ..mu..g/ml for B. subtilis DNA and 360 ..mu..g/ml for PBS2 DNA; the V/sub max/ value for denatured PBS2 DNA containing uracil is 7-fold greater than that for denatured B. subtilis DNA containing thymine. However, lower molecular weight DNAs have 10-fold lower apparent K/sub m/ values and show similar V/sub max/ values for both B. subtilis and PBS2 DNAs. Thus, the PBS2 phage-induced DNA polymerase (which likely replicates only uracil-containing phage DNA using dUTP in vivo) has little selectivity for uracil- versus thymine-containing deoxyribonucleotides or DNA in vitro.

• Research Organization: Univ. of Michigan, Ann Arbor
• OSTI ID: 6803743
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 253:23; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AZINES
BACILLUS
BACILLUS SUBTILIS
BACTERIA
BACTERIOPHAGES
BIOCHEMICAL REACTION KINETICS
DNA
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
KINETICS
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PARASITES
POLYMERASES
PURIFICATION
PYRIMIDINES
REACTION KINETICS
THYMINE
URACILS
VIRUSES