       Document 0199
 DOCN  M9580199
 TI    Insights into DNA polymerization mechanisms from structure and function
       analysis of HIV-1 reverse transcriptase.
 DT    9506
 AU    Patel PH; Jacobo-Molina A; Ding J; Tantillo C; Clark AD Jr; Raag R;
       Nanni RG; Hughes SH; Arnold E; Center for Advanced Biotechnology and
       Medicine, Rutgers; University, Piscataway, New Jersey 08854-5638, USA.
 SO    Biochemistry. 1995 Apr 25;34(16):5351-63. Unique Identifier : AIDSLINE
       MED/95244458
 AB    When the single-stranded RNA genome of HIV-1 is copied into
       double-stranded DNA, the viral enzyme reverse transcriptase (RT)
       catalyzes the addition of approximately 20,000 nucleotides; however, the
       precise mechanism of nucleotide addition is unknown. In this study, we
       attempt to integrate the genetic data and biochemical mechanism of DNA
       polymerization with the structure of HIV-1 RT complexed with a dsDNA
       template-primer. The first step of polymerization involves the physical
       association of a polymerase with its nucleic acid substrate. A
       comparison of the structures of HIV-1 RT in the presence and absence of
       DNA indicates that the tip of the p66 thumb moves approximately 30 A
       upon DNA binding. This conformational change permits numerous
       interactions between residues of alpha-helices H and I in the thumb
       subdomain and the DNA. Measurements of DNA binding affinity for nucleic
       acids with double-stranded DNAs that have an increasing number of bases
       in the template overhang and molecular modeling suggest that portions of
       beta 3 and beta 4 within the fingers subdomain bind single-stranded
       regions of the template. Measurements of nucleotide incorporation
       efficiency (kcat/Km) show that the binding and incorporation of the next
       complementary nucleotide are not dependent on the length of the template
       overhang. Molecular modeling of an incoming nucleotide triphosphate
       (dTTP), based in part on the position of mercury atoms in a
       RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary
       structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta
       10 determine the topology of the dNTP-binding site. These results also
       suggest that nucleotide incorporation is accompanied by a protein
       conformational change that positions the dNTP for nucleophilic attack.
       Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer
       strand could be metal-mediated, and Asp185 may be directly involved in
       stabilizing the transition state. The translocation step may be
       characterized by rotational as well as translational motions of HIV-1 RT
       relative to the DNA double helix. Some of the energy required for
       translocation could be provided by dNTP hydrolysis and could be coupled
       with conformational changes within the nucleic acid. A structural
       comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase
       identified regions within T7 RNA polymerase which are not present in the
       other two polymerases that might help this polymerase to remain bound
       with nucleic acids and contribute to the ability of the T7 RNA
       polymerase to polymerize processively.
 DE    Amino Acid Sequence  Base Sequence  Binding Sites  DNA Primers  *DNA
       Replication  DNA, Viral/BIOSYNTHESIS/CHEMISTRY  Genome, Viral
       HIV-1/*ENZYMOLOGY/GENETICS  Kinetics  Models, Molecular  Molecular
       Sequence Data  Nucleic Acid Conformation  *Protein Conformation  Protein
       Structure, Secondary  Reverse Transcriptase/*CHEMISTRY/*METABOLISM  RNA,
       Viral/GENETICS  Support, Non-U.S. Gov't  Support, U.S. Gov't, P.H.S.
       Templates  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

