BaciZZus subtilis Bacteriophage PBS2-induced DNA Polymerase

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 dlTP are incorporated efficiently as substrates and are competitive inhibitors at the same active site. The apparent K,,, and Ki values are about 6 pM for dYM’P and 15 pM for dUTP, when denatured, uracil-containing PBS2 DNA is used as template. The apparent K,,, values are lower with denatured, thyminecontaining B. subtilis or salmon sperm DNA (3.9 PM for dUTP and 2.6 PM for dlY’P). 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 Km values are 270 pg/ml for B. subtilis DNA and 360 pg/ml for PBS2 DNA; the V,, 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 lo-fold lower apparent K,,, values and show similar V,,,,, values for both B. subtilis and PBS2 DNAs. Thus, the PBS2 phageinduced DNA polymerase (which likely replicates only uracil-containing phage DNA using dUTP in ho) has little selectivity for uraciluersus thymine-containing deoxyribonucleotides or DNA in vitro.