Stimulation of HIV-1 minus strand strong stop DNA transfer by genomic sequences 3' of the primer binding site

The mechanism of human immunodeficiency virus 1 (HIV-1) minus strand transfer was examined using a genomic RNA sequence-based donor-acceptor template system. The donor RNA, D199, was a 199-nucleotide sequence from the 5'-end of the genome to the primer binding site (PBS) and shared 97 nucleotides of homology with the acceptor RNA. To investigate the influence of RNA structure on transfer, a second donor RNA, D520, was generated by extending the 3'-end of D199 to include an additional 321 nucleotides of the genome. The position of priming, length of homology with the acceptor, and length of cDNA synthesized were identical with the two donors. Interestingly, at 200% NC coating, donor D520 yielded a transfer efficiency of about 75% compared with about 35% with D199. A large proportion of the D520 promoted transfers occurred after the donor RNA was copied to the end. Analysis of donor RNA cleavage, the acceptor invasion site and R homology requirements indicated that transfers with D520 involved a similar but more efficient acceptor invasion mechanism compared with D199. RNA structure probing by RNase T1 and the RT pause profile during synthesis indicated conformational differences between D199 and D520 in the starting structure, and in dynamic structures formed during synthesis within the R region. Overall observations suggest that regions 3' of the primer binding site influence the conformation of the R region of D520 to facilitate steps that promote strand transfer.     

Kinetic analysis of Escherichia coli RNase H using DNA-RNA-DNA/DNA substrates.

The kinetic properties of Escherichia coli ribonuclease H (RNase H) were investigated using oligonucleotide substrates that consist of a short stretch of RNA, flanked on either side by DNA (DNA-RNA-DNA). In the presence of a complementary DNA strand, RNase H cleavage is restricted to the short ribonucleotide stretch of the DNA/RNA heteroduplex. The DNA-RNA-DNA substrate utilized for kinetic studies: (formula; see text) is cleaved at a single site (decreases) in the presence of a complementary DNA strand, to generate (dT)7-(rA)2-OH and p-(rA)2-(dT)9. Anion exchange high performance liquid chromatography was used to separate and quantitate the cleavage products. Under these conditions, RNase H-specific and nonspecific degradation products could be resolved. Kinetic parameters were measured under conditions of 100% hybrid formation (1.2-1.5 molar excess of complementary DNA, T much less than Tm). A linear double reciprocal plot was obtained, yielding a Km of 4.2 microM and a turnover number of 7.1 cleavages per s per RNase H monomer. The kinetic properties of substrate analogs containing varying lengths of RNA (n = 3-5) and 2'-O-methyl modifications were also investigated. Maximal turnover was observed with DNA-RNA-DNA substrates containing a minimum of four RNA residues. Kcat for the rA3 derivative was decreased by more than 100-fold. The Km appeared to decrease with the size of the internal RNA stretch (n = 3-5). No significant difference in turnover number of Km was observed when the flanking DNA was replaced with 2'-O-methyl RNA, suggesting that RNase H does not interact with this region of the heteroduplex.