DNA primase. Processivity and the primase to polymerase alpha activity switch.
Calf thymus DNA primase was examined to determine the kinetic parameters that define its unusual processivity. At 37 degrees C, the major products were 8-9 and 2-3 nucleotides long. The 2-mer was the predominant product when considered on a molar basis. At each polymerization cycle en route to synthesis of a unit length primer (7-10 nucleotides), processivity was defined by competition of enzyme dissociation with ATP binding as well as an ATP independent step(s). Reducing the temperature to 25 degrees C had relatively little effect on the production of primers less than or equal to 6 nucleotides long, but greatly enhanced production of primers twice (16-18 nucleotides) the normal unit length. Kinetic analysis revealed that synthesis of these longer primers largely involves dissociation of the primase after completion of the unit length primer. After synthesis of a primer, the primase-polymerase complex normally switches to polymerase activity. Only primers greater than or equal to 7 nucleotides long were utilized by the polymerase regardless of the dNTP concentration, indicating that the signal for the primase to polymerase activity switch is primer completion. During the switch, either the primer-template does not dissociate from the complex or the complex has extraordinarily high affinity for the primers. At 25 degrees C and physiological dNTP concentrations the activity switch is very efficient, greater than 90% of the primers are elongated. However, at 37 degrees C the switch is much less efficient, likely due to primer-template denaturation.
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
R D
RD
Kuchta
B
B
Reid
L M
LM
Chang
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004247:DNA / Q000302:isolation & purification; D004257:DNA Polymerase II / Q000378:metabolism*; D019915:DNA Primase; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology
D000255:Adenosine Triphosphate; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D003850:Deoxyribonuclease I