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

Abstract

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.

Figure 1

Reaction temperature optima of RTs. The relative RNA-directed DNA polymerase activities of H+ M-MLV RT (inverted triangle), H– M-MLV RT (triangle), H+ AMV RT (open circle) and H– AMV RT (filled circle) were measured (Materials and Methods) at the temperatures indicated. The optima (middle of the temperature range at which the relative activity was >0.9) are indicated by broken vertical lines.

Figure 2

Processivity measurements of ASLV RTs and H+ M-MLV RT. DNA was synthesized in the presence (upper panel) and absence (lower panel) of a heparin trap from RSV cRNA annealed to a 5′ ³²P-labeled DNA 20mer (Materials and Methods). Reaction mixtures were incubated for 0.5 and 2 min and contained cloned H+ AMV RT (A), native AMV RT (B), H– AMV RT (C), cloned H+ RSV RT (D), H– RSV RT (E) or H+ M-MLV RT (G). (F, upper panel) The total inhibition of cDNA synthesis by cloned H+ AMV RT initiated in the presence of both template-primer and heparin, demonstrating the effectiveness of the heparin trap. ³²P-labeled markers were 1 kb DNA ladder (lane M1) and 100 bp DNA ladder (lane M2). The arrow indicates full-length product.

Figure 3

Deadenylation of CAT cRNA•p(dT)_12–18 by AMV RT. CAT cRNA ³²P-labeled at the 3′ end and annealed to p(dT)_12–18 was incubated with H+ and H– AMV RT for various times at 37 or 55°C and then fractionated on a denaturing 20% polyacrylamide gel (Materials and Methods). Template-primer that was not incubated is shown in lane A. Samples were incubated at 4°C for 2 min without RT (lane B); 55°C for 2 min without RT (lane C); 55°C for 15 s (lane D) and 2 min (lane E) with 0.42 pmol H+ AMV RT; 37°C for 5 min without RT (lane F); 37°C for 5 min with 4.2 pmol H+ AMV RT (lane G); 55°C for 15 s (lane H) and 2 min (lane I) with 0.42 pmol H– AMV RT; and 37°C for 5 min with 1 U E.coli RNase H (lane J). ³²P-labeled markers were 10 bp ladder, (dT)₄ and (dT)₆.

Figure 4

The effect of temperature on full-length cDNA synthesis from equimolar amounts of cRNAs. cDNAs synthesized by H– and H+ M-MLV RT (400 nM) at 42°C (lane A), 45°C (lane B), 48°C (lane C), 50°C (lane D) and 52°C (lane E) from equimolar amounts (12 nM each) of cRNAs of 1.4 (0.1 µg), 2.4 (0.17 µg), 4.4 (0.31 µg), 7.5 (0.54 µg) and 9.5 kb (0.67 µg) were analyzed by alkaline agarose gel electrophoresis (Materials and Methods). Incubation time was 30 min. ³²P-labeled DNA (1 kb ladder) was run as a marker (lane M).