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Nucleic Acids Research, 2002, Vol. 30, No. 14 3118-3129
© 2002 Oxford University Press

The role of template-primer in protection of reverse transcriptase from thermal inactivation

Gary F. Gerard*, R. Jason Potter, Michael D. Smith, Kim Rosenthal, Gulshan Dhariwal, Jun Lee and Deb. K. Chatterjee

Invitrogen Corporation, 7335 Executive Way, Frederick, MD 21704, USA

*To whom correspondence should be addressed. Tel: +1 240 379 4728; Fax: +1 240 379 4333; Email: gary.gerard{at}invitrogen.com

We compared the thermal stabilities of wild-type recombinant avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) with those of mutants of the recombinant enzymes lacking RNase H activity. They differed in resistance to thermal inactivation at elevated temperatures in the presence of an RNA/DNA template-primer. RNase H-minus RTs retained the ability to efficiently synthesize cDNA at much higher temperatures. We show that the structure of the template-primer has a critical bearing on protection of RT from thermal inactivation. RT RNase H activity rapidly alters the structure of the template-primer to forms less tightly bound by RT and thus less able to protect the enzyme at elevated temperatures. We also found that when comparing wild-type or mutant AMV RT with the respective M-MLV RT, the avian enzymes retained more DNA synthetic activity at elevated temperatures than murine RTs. Enzyme, template-primer interaction again played the most significant role in producing these differences. AMV RT binds much tighter to template- primer and has a much greater tendency to remain bound during cDNA synthesis than M-MLV RT and therefore is better protected from heat inactivation.


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