DNA & RNA SYNTHESIS IN VITRO TECHNOLOGY

DNA & RNA Synthesis in Vitro Technology

Abstract

In vitro DNA synthesis on single-stranded circular DNA can be initiated by RNA primers. RNA chains are covalently extended by DNA polymerase II from KB cells and DNA polymerase I from Micrococcus luteus, but not by an RNA-dependent DNA polymerase from avian myeloblastosis virus. The reaction product consists of DNA chains with a piece of RNA at their 5'-ends, hydrogen bonded to the template DNA. The primer RNA is linked to the product DNA via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H. The possible role of ribonuclease H in RNA-primed DNA synthesis in vivo is discussed.

DNA & RNA Synthesis in Vitro Technology

Introduction

Genetic or biochemical evidence for direct participation of RNA synthesis in DNA replication has recently been reported for Escherichia coli (1, 2), phage lambda (3), and phage M13 (4). In each instance, the basic observation was that lack of or inhibition of transcription of certain regions of the genome prevented DNA replication. In what way transcription is involved in DNA synthesis is not known. In the case of phage M13 DNA synthesis, the suggestion has been made that a small piece of RNA, synthesized by DNA-dependent RNA polymerase on the parental plus-strand DNA, promotes the initiation of minus-strand synthesis by acting as primer for DNA polymerase (4).

The occurrence of such RNA-primed DNA synthesis within higher cells may well be the reason for the widespread existence of ribonuclease H (RNase H) (5). This enzyme, now under active study in this laboratory, specifically degrades RNA hybridized to DNA, and thus is capable of removing 5'-terminal RNA primers from DNA chains if a complementary DNA strand is present. Thus, we looked at whether the mammalian DNA polymerase II from KB cells would work better if concomitant RNA synthesis was allowed. We obtained positive results; other workers (6, 7) independently reported similar observations with other systems. In our in vitro experiments, mammalian DNA polymerase II and Micrococcus luteus DNA polymerase synthesize DNA complementary to single-stranded circular OX DNA by covalent extension, either of preformed RNA chains annealed to the template DNA (uncoupled system), or of RNA chains generated by addition of RNA polymerase to the reaction mixtures (coupled system). The RNA part of the resulting RNA-DNA chains can be specifically removed by RNase H.

Main Part

Stimulation of DNA Synthesis by Concomitant RNA Synthesis

Mammalian DNA polymerase II is inactive with singlestranded circular DNA as template. Like all known DNA polymerases, it requires a priming polynucleotide with a free 3'-hydroxyl group for initiation. (8) However, when DNAdependent RNA polymerase (E. coli) and ribonucleoside triphosphates were added to reaction mixtures containing ,X DNA, KB DNA polymerase II, and deoxyribonucleoside triphosphates, DNA synthesis occurred (Table 1). That this DNA synthesis required concomitant RNA synthesis is shown by the results of double-label experiments (Table 1), in which the inhibition of RNA synthesis by addition of rifampicin or omission of ribonucleoside triphosphates reduced the stimulatory effect of RNA polymerase on DNA synthesis ...