Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: Functional implications for template-primer binding to the fingers domain

Reverse transcriptase (RT) serves as the replicative polymerase for retrovi ruses by using RNA and DNA-directed DNA polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the s ingle-stranded RNA genome. In an effort to obtain detailed structural infor mation about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 Angstrom resolution of an N-terminal f ragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the f ingers and palm domains from Moloney murine leukemia virus reverse transcri ptase. We have also determined the crystal structure at 3.0 Angstrom resolu tion of the fragment complexed to DNA with a single-stranded template overh ang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA ato ms involved in the interactions include the 3'-OH group from the primer str and and minor groove base atoms and sugar atoms from the n - 2 and n - 3 po sitions of the template strand, where n is the template base that would pai r with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residu es in the beta 4-beta 5 loop (beta 3-beta 4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to H IV-1 RT but related in the types of interactions formed between protein and DNA. Ln addition, the DNA in all of these complexes is bound in the same c left of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and hav e characterized the resulting enzymes. We now propose that nucleic acid bin ding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process. (C) 2000 Academic Press.