Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis.
The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5'-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 degrees C was 33 nucleotide residues, due to the chain-terminating action of 2',3'-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5'-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5'-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5'-end released unlabeled DNA with a free 5'-terminus and 32P-labeled ribonucleoside 3',5'-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3'-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3'-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.
D001412:Bacillus subtilis / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000302:isolation & purification; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000032:analysis; D054306:Dideoxynucleotides; D006868:Hydrolysis; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D012329:RNA, Bacterial; D013997:Time Factors
C037771:5-mercurideoxycytidine triphosphate; D004269:DNA, Bacterial; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; D012329:RNA, Bacterial; C037773:2',3'-dideoxyadenosine triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010727:Phosphoric Diester Hydrolases