Products of a lagging DNA strand synthesis of nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA.
Nucleoprotein (NP) complexes present in selected fractions of the separated three basic components A, B, C of the postmicrosomal sediment (POMS) (Ríman and Sulová, 1997a) were used as a source of nucleic acid synthesizing activities (NA-SAs) expressed in reactions in vitro. These were performed in the absence and presence of N2-(p-butylphenyl) deoxyguanosine 5'-triphosphate (BuPdGTP), aphidicolin (Aph) and carbonyldiphosphonate (COMDP), inhibitors allowing differentiation between two DNA polymerases (pols) involved in a lagging DNA strand synthesis (LSS). Reaction products were isolated and analyzed by polyacrylamide gel electrophoresis (PAGE) in denaturing conditions. Products labelled with [alpha-32P]dAMP or [alpha-32P]AMP were represented by intermediates significant for LSS. Okazaki fragment precursors, whose synthesis was inhibited by BuPdGTP and resistant to Aph, and whose radioactive RNA label was DNase I-sensitive, represented products formed in vitro by NP complexes of POMS component C. Okazaki fragments 127 b in length, whose synthesis was insensitive to BuPdGTP but inhibited by Aph and COMDP, were characteristic of the reactions accomplished by NP complexes of POMS component B while products of NA-SAs of NP complexes of POMS component A were represented by Okazaki fragments up to 280 b in length, whose synthesis was most sensitive to Aph. In accord with previous data (Ríman and Sulová, 1997b), COMDP strongly stimulated production of RNAs corresponding in length with initiator RNAs (iRNAs). However, in dependence on reaction conditions also ribodeoxyribonucleotide primers can be produced by NP complexes of POMS component C, suggesting the occurrence of two primase (Pr) catalytic modes influenced by dNTP/rNTP relation and thus, by COMDP, a strong competitor for dNTPs. These results represent the first direct evidence that an extrachromosomal DNA organized into special NP complexes can be replicated extrachromosomally by a mechanism of LSS.
Ríman
J
J
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Sulová
A
A
D016590:Aphidicolin / Q000494:pharmacology; D009189:Avian Myeloblastosis Virus / Q000235:genetics*; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors; D004279:DNA, Viral / Q000096:biosynthesis*; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D004164:Diphosphonates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D009698:Nucleoproteins / Q000378:metabolism*; D014758:Viral Core Proteins / Q000378:metabolism
D004279:DNA, Viral; D003848:Deoxyguanine Nucleotides; D004164:Diphosphonates; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D009698:Nucleoproteins; D014758:Viral Core Proteins; C062324:carbonyldiphosphonate; D016590:Aphidicolin; C040364:N(2)-(4-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate; D004257:DNA Polymerase II