Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A.
DNA primase has been partially purified from wheat germ. This enzyme, like DNA primases characterized from many procaryotic and eucaryotic sources, catalyses the synthesis of primers involved in DNA replication. However, the wheat enzyme differs from animal DNA primase in that it is found partially associated with a DNA polymerase which differs greatly from DNA polymerase alpha. Moreover, the only wheat DNA polymerase able to initiate on a natural or synthetic RNA primer is DNA polymerase A. In this report we describe in greater detail the chromatographic behaviour of wheat DNA primase and its copurification with DNA polymerase A. Some biochemical properties of wheat DNA primase such as pH optimum, Mn + 2 or Mg + 2 optima, and temperature optimum have been determined. The enzyme is strongly inhibited by KCI, cordycepine triphosphate and dATP, and to a lesser extent by cAMP and formycine triphosphate. The primase product reaction is resistant to DNAse digestion and sensitive to RNAse digestion. Primase catalyses primer synthesis on M13 ssDNA as template allowing E.coli DNA polymerase I to replicate the primed M13 single-stranded DNA leading to double-stranded M13 DNA (RF). M13 replication experiments were performed with wheat DNA polymerases A, B, CI and CII purified in our laboratory. Only DNA polymerase A is able to recognize RNA-primed M13 ssDNA.
Laquel
P
P
Laboratoire de Biologie Moléculaire Végétale, IBCN-CNRS, Bordeaux, France.
Castroviejo
M
M
Litvak
S
S
D002848:Chromatography, DEAE-Cellulose; D000242:Cyclic AMP / Q000494:pharmacology; D004258:DNA Polymerase III / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005573:Formycins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D008345:Manganese; D011189:Potassium Chloride / Q000494:pharmacology; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology; D013696:Temperature; D014908:Triticum / Q000201:enzymology*
D003838:Deoxyadenine Nucleotides; D005573:Formycins; D012265:Ribonucleotides; C032552:formycin triphosphate; D008345:Manganese; C011746:3'-deoxyadenosine 5'-triphosphate; D011189:Potassium Chloride; D000242:Cyclic AMP; D019915:DNA Primase; C065639:DNA polymerase A; D012316:RNA Nucleotidyltransferases; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium