Transcriptional Role in Deoxyribonucleic Acid Replication

• Published in: The Journal of biological chemistry 1973-12, Vol.248 (24), p.8465-8473
• Main Author: Roychoudhury, Ranajit
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)43156-6

DNA synthesis in an in vitro system proceeds by a covalent chain extension of the newly generated primers in the presence of Mg2+, Escherichia coli DNA polymerase I or its large fragment, ribo- and deoxyribonucleoside triphosphates, RNA polymerase, and either single-stranded fd DNA or d(T)1000 as template. The synthesis is strongly influenced by the number of RNA chains and the kinetics of polymerization is nonlinear, showing a brief lag followed by a period of rapid synthesis before reaching a saturation level. However, a linear kinetics is observed when the number of new chain initiation is controlled (i.e. rifampicin added 1 to 5 min after RNA synthesis). A degradation of ribo-strands is observed during DNA synthesis. When d(T)1000 fully transcribed with RNA polymerase (1:1 hybrid) is employed as template-primer, both the whole enzyme and the large fragment of DNA polymerase I catalyze incorporation of dAMP residues. During the initial synthetic period there is a mole for mole degradation of ribo-strands by the whole enzyme. With large fragment DNA chain growth proceeds by a process of of strand displacement. In the initial synthetic period the 5′→3′ exonuclease activity of DNA polymerase I attacks the 5′ ends of the ribostrands endonucleolytically liberating 5′-triphosphate-terminated di- or oligonucleotides. Subsequent action of the nuclease activity proceeds exonucleolytically liberating 5′-mononucleotides as the DNA chain growth proceeds. The results suggest that the 5′→3′ exonuclease activity of DNA polymerase is capable of removing RNA primers from the final product of replication in vivo.