The Role of Deoxyribonucleic Acid in Ribonucleic Acid Synthesis

X. THE INFLUENCE OF 6-METHYLAMINOPURINE ON NUCLEIC ACID SYNTHESIS IN VITRO

From the ¹ From the Department of Molecular Biology, Albert Einstein College of Medicine, New York, New York 10461

The methylated adenine derivatives, 6-methylaminopurine ribonucleoside triphosphate (6MeATP), -³²P-6-dimethylaminopurine ribonucleoside triphosphate, (-³²P-6diMeATP) and 6-methylaminopurine deoxynucleoside triphosphate (d6MeATP) have been synthesized. These compounds were tested as substrates with the ribonucleic acid polymerase and deoxyribonucleic acid polymerase systems. In the presence of deoxyadenylate-deoxythymidylate copolymer or calf thymus DNA as primers in the DNA polymerase system, evidence of incorporation of d6MeATP was obtained. This reaction occurred at a rate approximately 65% of that observed with deoxyadenosine triphosphate. In RNA synthesis catalyzed by RNA polymerase, 6MeATP replaced ATP whereas -³²P-6diMeATP was completely inactive as a substrate. RNA synthesis with 6MeATP occurred at a rate approximately 5% of that observed with ATP. It was also established that labeled RNA synthesized in the presence of -³²P-6MeATP contained 6-methylaminopurine ribonucleoside phosphate-³²P. The latter compound was isolated after venom phosphodiesterase degradation of RNA as the only labeled nucleotide present.

The low rate of RNA synthesis with 6MeATP as substrate can be attributed to two factors. The apparent affinity constant of 6MeATP was 5.6 x 10^-4 m whereas the apparent affinity constant of ATP for RNA polymerase was 6.3 x 10^-5 m. The second factor that seemed to contribute to the low activity of RNA synthesis with 6MeATP is the relatively poor hydrogen bonding formed between 6-methylaminopurine and thymine of the priming DNA. Thus, polyriboadenylate formation catalyzed by RNA polymerase occurred only with ATP and was not detected when 6MeATP replaced ATP. A mixed polymer containing 6MeAMP and AMP was formed only when the RNA polymerase-catalyzed reaction was carried out with a mixture of 6MeATP and ATP.

Other enzyme systems have been shown to utilize 6MeATP. The compound replaced ATP in the hexokinase reaction as well as with the RNA-dependent polyriboadenylate polymerase. The effect of the presence or absence of 6-methylaminopurine in T2 DNA and T4 DNA on the enzymatic synthesis of RNA was also examined. No differences in the rate or yield of RNA synthesis were found. In addition, the influence of the presence or absence of 6-methylaminopurine in DNA on RNA chain initiation was also checked and no differences were noted.

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