Abstract
Single-stranded DNA binding protein is a key component in growth of bacteriophage T7. In addition, DNA synthesis by the purified in vitro replication system is markedly stimulated when the DNA template is coated with Escherichia coli single-stranded DNA binding protein (SSB). In an attempt to understand the mechanism for this stimulation, we have studied the effect of E. coli SSB on DNA synthesis by the T7 DNA polymerase using a primed single-stranded M13 DNA template which serves as a model for T7 lagging strand DNA synthesis. Polyacrylamide gel analysis of the DNA product synthesized on this template in the absence of SSB indicated that the T7 DNA polymerase pauses at many specific sites, some stronger than others. By comparing the position of pausing with the DNA sequence of this region and by using a DNA template that contains an extremely stable hairpin structure, it was found that many, but not all, of these pause positions correspond to regions of potential secondary structure. The presence of SSB during synthesis resulted in a large reduction in the frequency of hesitations at many sites that correspond to these secondary structures. However, the facts that a large percentage of the pause sites remain unaffected even at saturating levels of SSB and that SSB stimulates synthesis on a singly primed poly(dA) template suggested that other mechanisms also contribute to the stimulation of DNA synthesis caused by SSB. Using a sucrose gradient analysis, we found that SSB increases the affinity of the polymerase for single-stranded DNA that this increased binding is only noticed when the polymerase concentration is limiting. The effect of this difference in polymerase affinity was clearly observed by a polyacrylamide gel analysis of the product DNA synthesized during a limited DNA synthesis reaction using conditions where only two nucleotides are added to the primer. Under these circumstances, where the presence of hairpin structures should not contribute to the stimulatory effect of SSB, we found that the extension of the primer is stimulated 4-fold if the DNA template is coated with SSB. Furthermore, SSB had no effect on this synthesis at large polymerase to template ratios.
Highlights
Tem is markedly stimulatedwhen the DNA template is phages which infect E. coli [4]
DNA product synthesized on this template in the absence of SSB indicated that the T7 DNA polymerase synthesis by purified DNA polymerases;T7 DNA polymerase, E . coli DNA polymerase 11, and DNA polymerase I11 holoenpauses at many specific sites, some stronger than oth- zyme are stimulated by SSB, while little stimulation is obers
SSB has been shown t o increase the template that contains an extremelystablehairpin fidelity of DNA synthesisby a wide range of DNA polymerases structure, it wasfound that many, but not all, of these [14, 15]
Summary
Polyacrylumide Gel Electrophoresis-DNA synthesis reactions (50 p l ) were carried out as described above except that the dTTP was unlabeled and the pentadecamer primer was labeled at the 5' end with 32P(1-4 X 10, cpm/pmol). T7 DNA polymerase activity was assayed by adding 20 pl of the indicated fraction to a reaction containing denatured salmon sperm DNA (167 pg/ml), 87 mM KPO, (pH 7.4), 6.7 mM MgC12, 5 mM mercaptoethanol, 100 p~ dATP, dCTP, dGTP, and [CY-'~P]~T(T6P0 cpm/pmol). Reactions (100 pl) were incubated at 37 "C for 30 min and terminated by the addition of 10 p1 of 0.5 M EDTA (pH 8).M13mp9 ["]DNA was analyzed by adding 10O-pl aliquots of the gradient fractions to Beckman ReadysolvMP aqueous scintillation fluid (10 ml) and counting on a Beckman LS 7500 liquid scintillation counter.
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