Abstract
A previous paper reported the purification (from mouse cell extracts) and some of the properties of a protein, alpha accessory factor (AAF), that specifically stimulates DNA polymerase alpha/primase (1). We describe here studies on the mechanism of action of AAF. In the presence of AAF and a large excess of single-stranded circular DNA template, a molecule of DNA polymerase alpha/primase interacts with a single template DNA molecule priming and synthesizing multiple short DNA fragments covering thousands of nucleotides without detaching from the template, and, by many-fold repetition of the process, accomplishes serial replication of the population of DNA molecules. In contrast, without AAF the reaction involves the whole population of DNA molecules in parallel and with a very large number of binding events between DNA polymerase alpha/primase and DNA [corrected] template. The profound [corrected] increase in affinity of DNA polymerase alpha/primase for the DNA template that characterizes the mechanism suggests a functional identification of AAF as a template affinity protein. The resulting greater efficiency accounts for the ability of AAF to stimulate both the primase and polymerase activities of DNA polymerase alpha/primase. AAF also increases the processivity of DNA polymerase alpha/primase from approximately 15 to approximately 115 nucleotides, a size similar to that of mammalian Okazaki fragments, and it appears to allow DNA polymerase alpha/primase to traverse double-stranded regions of a DNA template. These features of the mechanism of AAF suggest that it may have a role in assisting DNA polymerase alpha/primase in synthesis of the lagging strand of a replication fork.
Highlights
A previous paper reported the purification and some of the properties of a protein, alpha accessory factor (AAF), that stimulates DNA polymerase u/primase [1]
Kinetics of this reaction were tested in the reaction of DNA polymerase cY/primase with single-stranded circular DNA in the presence of AAF to see if there was an effect from the order of addition of components or a lag, indicating formation of a complex (Fig. 1)
The stimulatory effect of AAF can be seen from the results of a series of reactions in which DNA polymerase cY/primase and AAF were kept constant while the amounts of singlestranded circular DNA were varied (Table I)
Summary
DNA (50 pg) was digested with pancreatic DNase (0.6 pg) for 30 min at 37 “C ina volumeof 0.1 ml (i0 mM Tris(Cl-), pH 8.1;20 mM KCI,. In length on neutral agarose gel electrophoresis. The annealed mixture was passed through a lo-ml bed column of Sephacry S-200 in 10 mM Tris(Cl-), pH 7.6,l mM EDTA, and the fractions containing circles with annealed fragments, which were in the void volume (monitored by Az6,, and neutral agarose gel electrophoresis), were pooled. Unless stated otherwise the reaction mixtures were 20 ~1 and contained 50 mM Tris(Cl-), pH 7.6, 50 mM potassium acetate, 8 mM magnesium acetate, 2 mM dithiothreitol, 0.1 mM EDTA, 2 mM rATP, 0.2 mM each of rCTP, rGTP, and rUTP, 50 pM each of dATP, dCTP, dGTP, and dTTP, 0.1 mg/ml bovine plasma albumin, 0.01% Nonidet. Reactions were stopped by transferring the tubes back to the ice bath and adding l/9 volume of. Photofluorographs were made by photographing (Polaroid 667 film, Wratten 23A filter) ethidium bromide containing neutral agarose gels transilluminated with ultraviolet (300 nm) light
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