The mechanism of action of an accessory protein for DNA polymerase alpha/primase.
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.
Goulian
M
M
Department of Medicine, University of California, San Diego, La Jolla 92093-0613.
Heard
C J
CJ
D000818:Animals; D019915:DNA Primase; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D051379:Mice; D011506:Proteins / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis / Q000378:metabolism*; D013698:Templates, Genetic
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D011506:Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases