Endogenous RNasa-sensitive DNA polymerase complex from rat tissues : characterization of the reaction and the products, and of the DNA polymerases released by RNase-treatment

Moranelli, Francesco (1977) Endogenous RNasa-sensitive DNA polymerase complex from rat tissues : characterization of the reaction and the products, and of the DNA polymerases released by RNase-treatment. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

An endogenously-templated DNA polymerase activity from rat thymus and liver has been partially purified and characterized, and the product of the reaction analyzed. The enzyme from both sources was shown to be sensitive to pretreatment with RNases [hence it is referred to as a RNase-sensitive DNA polymerase (RS-DP)]. The molecular weight of the RS-DP complex, estimated from Sepharose 6B gel filtration, is 280,000 daltons. -- The RNA associated with the RS-DP is probably single-stranded (and therefore functions as a template) since the activity remained sensitive to RNase-treatment under conditions in which only single-stranded RNA is digested. The putative RNA template is heteropolymeric in nature, since all four nucleotides were incorporated into the DNA product to a similar extent (also indicating that the enzyme is not simply a terminal transferase). The enzyme is probably not of viral origin, as the activity was not stimulated by non-ionic detergents and also had a buoyant density (1.05 g/cm³) in a sucrose gradient which differed greatly from that reported for the type-C viral activity (1.22-1.24 g/cm³). Inhibition studies with actinomycin D and distamycin A, however, revealed a similarity to the viral RNA-directed DNA polymerase (RD-DP), supporting the notion that the RNA in the RS-DP complex functions as a template. Additional supporting evidence for a template function for the RNA derives from buoyant density analysis of the product of the rat liver RS-DP activity. -- The RS-DP activity differs from DNA-directed DNA polymerases in its preference for MN²⁺ to Mg²⁺ as the divalent cofactor. Furthermore, the enzyme is not inhibited by N-ethylmaleimide and also responds differently to heparin and polyamines than does the rat thymus DNA polymerase α. -- Two lower molecular weight DNA polymerases (70,000 and 30-40,000 daltons, respectively denoted as Peaks II and III), were derived from the RS-DP complex by extensive RNase-treatment. These activities are probably not proteolytic fragments of a higher molecular weight DNA polymerase, since RNase-treatment in the presence of the protease inhibitors Trasylol and phenylmethylsulfonyl fluoride also resulted in the release of these activities. -- The Peak II and III activities differ from each other in a number of their properties, and furthermore, differ from other eukaryotic DNA polymerases described in the literature, indicating that the enzymes are distinct and probably unique DNA polymerases.

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