Influence of gap size on 5′ end-directed cleavage of Md1 by M-MuLV reverse transcriptase. 5′ end-labeled Md1 was used as a substrate without upstream RNA (No Upstream), as a substrate with PPT20 annealed immediately upstream (Nick), or as substrates with gaps of 1 to 5 bases between the 3′ terminus of upstream PPT20 and the 5′ end of Md1 (1 to 5 b Gap). Substrates were incubated with M-MuLV reverse transcriptase, and analyzed as described in Fig. 2. In Md1, cleavage site A is +2/+3, site B is +5/+6, and the other sites are as described in Fig. 3.