DNA primase from KB cells. Evidence for a novel model of primase catalysis by a highly purified primase/polymerase-alpha complex.
The oligonucleotide primers synthesized by a highly purified KB fraction containing DNA primase and DNA polymerase-alpha activities display reproducible alterations of structure and composition in response to dNTPs. These observations are sufficiently explained by a novel model of primase catalysis that defines the primase as a complex enzyme with distinct catalytic properties that are regulated by mechanisms exquisitely sensitive to dNTP concentration. The enzyme performs the template-directed, de novo synthesis of 1 unit of oligoribonucleotide (canonical priming reaction) and then elongates that moiety to a limited extent by several cycles of addition of short tracts of homogeneous oligoribonucleotide or oligodeoxynucleotide. The oligomeric units have modal lengths of approximately 11- to 14- nucleotides that are postulated to reflect the inherent processivity of the catalytic mechanisms. Elongation is accomplished by two catalytic centers, or conformers of a single center, that are synchronously coupled, mutually exclusive, and extremely stringent for respective rNTP and dNTP substrates. Transitions between these two catalytic modes are regulated by dNTPs and demonstrable at dNTP:rNTP concentrations of less than or equal to 10(-4). In the absence of dNTPs, the primase produces a family of oligoribonucleotides, approximately 24- to 36- nucleotides long; at dNTP levels between 0.08 and 0.80 micron, the primase synthesizes mixed oligomers composed of strictly alternating tandem arrays of oligoribo- and oligodeoxynucleotide units; and at dNTP levels greater than or equal to 4.8 micron, the primase becomes stabilized in the deoxy mode after the initial oligoribo leads to oligodeoxy transition and products contain only a single 5' -terminal unit of oligoribonucleotide. The model predicts that the physiologically significant primer for DNA polymerase-alpha is a mixed 5' -oligoribo-3' -oligodeoxynucleotide and the signal which governs the switch from RNA leads to DNA synthesis is intrinsic in the primase mechanism and is generated by ambient dNTPs.
D002277:Carcinoma; D002460:Cell Line; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D006801:Humans; D007700:Kinetics; D009062:Mouth Neoplasms; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*
D009097:Multienzyme Complexes; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II