A DNA primase from mouse cells. Purification and partial characterization.
An enzymatic activity that synthesizes oligoribonucleotides in lengths of 9-10 nucleotides and multiples thereof has been purified over 10,000-fold from mouse hybridoma cells. The oligoribonucleotides serve as primers to initiate DNA synthesis, and the activity has properties expected of mammalian DNA primase. The most highly purified fraction has two major protein components, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, of 56,000 and 46,000 Da. These proteins co-purify in a 1:1 stoichiometry along with oligoribonucleotide synthesis activity and with an activity that initiates the synthesis of DNA by DNA polymerase alpha. The sedimentation coefficient on glycerol gradients is 5.5 S, and this is consistent with one 56,000- and one 46,000-Da subunit/native enzyme. No DNA polymerase activity was detected in the most highly purified fraction. Poly(dIT) is the most active template, whereas a variety of single-stranded DNA templates are 10-15% as active and double-stranded DNA templates are 10-15% as active and double-stranded DNA is less than 1%. rATP is an absolute requirement as is Mg2+. No ATPase activity was detected with or without addition of DNA, single- or double-stranded. (NH4)2SO4 and NaPO4 buffer, pH 7.6, are inhibitory above 20 mM, whereas KCl is inhibitory above 80 mM. beta-D-arabinose-CTP is a strong inhibitor of primase; approximately 50% inhibition is observed when present at one-fifth the concentration of rCTP.
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019915:DNA Primase; D006825:Hybridomas / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D051379:Mice; D008970:Molecular Weight; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification*; D013698:Templates, Genetic
D011089:Polydeoxyribonucleotides; D015636:Magnesium Chloride; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D008274:Magnesium