Mechanism of Action of the Endonuclease Associated with the αβ and ββ Forms of Avian RNA Tumor Virus Reverse Transcriptase

Abstract

Preparations of the αβ and the ββ forms of reverse transcriptase from the Prague C strain of Rous sarcoma virus grown in chicken embryo fibroblasts, the αβ and the ββ forms of the enzyme from the B77 strain of Rous sarcoma virus grown in duck embryo fibroblasts, and the αβ form of reverse transcriptase from avian myeloblastosis virus have been analyzed. All these enzyme preparations contain a Mn²⁺ -activated endonuclease activity. The ββ form of enzyme, in addition, contains a Mg²⁺ -dependent endonuclease. Such an activity is barely detectable in the αβ form of enzymes. The endonuclease associated with reverse transcriptase introduces single- and double-strand breaks containing 3′ OH and 5′ P termini into RF I DNA. The conversion of RF I DNA to RF III DNA is more readily catalyzed by the ββ form of reverse transcriptase. In contrast to a recently published report by Hizi et al. (J. Virol 41:974-981, 1982), we have failed to detect the conversion of RF I DNA to covalently closed relaxed circles (RF IV DNA) by any of the αβ form of enzymes tested. RF IV DNA was not produced by the ββ form of reverse transcriptase either. We conclude that topoisomerization is not an intrinsic activity of reverse transcriptase. Although the conversion of RF I DNA to RF II DNA was found to be rapid, the endonuclease associated with reverse transcriptase acted slowly on RF II, RF III, and RF IV DNAs. Circular and linear single-stranded DNAs were also susceptible to cleavage by the endonuclease at a rate comparable to nicking of RF I DNA. This pattern of activity suggests that the endonuclease cleaves the RF I DNA in the single-stranded regions of the DNA induced by its supercoiling. The preference of the αβ and the ββ forms of the endonuclease for viral DNA was tested with Rous-associated virus type 2 and Rous sarcoma virus transformation-defective Schmidt-Ruppin B strain DNA molecularly cloned in plasmid pBR322 and M13 DNA vectors, respectively. The rate of nicking of RF I DNA containing viral DNA or partial sequences of viral DNA with one or two tandem long terminal repeats was the same as when these sequences were not present in the host vectors. A similar lack of preference was observed with single-stranded M13 DNAs. Images