A 3.5 angstrom resolution electron density map of the HIV-1 reverse transcriptase heterodimer complexed with nevirapine, a drug with potential for treatment of AIDS, reveals an asymmetric dimer. The polymerase (pol) domain of the 66-kilodalton subunit has a large cleft analogous to that of the Klenow fragment of Escherichia coli DNA polymerase I. However, the 51-kilodalton subunit of identical sequence has no such cleft because the four subdomains of the pol domain occupy completely different relative positions. Two of the four pol subdomains appear to be structurally related to subdomains of the Klenow fragment, including one containing the catalytic site. The subdomain that appears likely to bind the template strand at the pol active site has a different structure in the two polymerases. Duplex A-form RNA-DNA hybrid can be model-built into the cleft that runs between the ribonuclease H and pol active sites. Nevirapine is almost completely buried in a pocket near but not overlapping with the pol active site. Residues whose mutation results in drug resistance have been approximately located.

The dipyridodiazepinone Nevirapine is a potent and highly specific inhibitor of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). It is a member of an important class of nonnucleoside drugs that appear to share part or all of the same binding site on the enzyme but are susceptible to a variety of spontaneous drugresistance mutations. The co-crystal-structure of HIV-1 RT

The reverse transcriptase from human immunodeficiency virus type 1 is a heterodimer consisting of one 66-kDa and one 51-kDa subunit. The p66 subunit Perhaps the most surprising aspect of the structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the observation that the polymerase domain assumes a different structure in the two subunits in spite of having the same polypeptide chain sequence (1). HIV-1 RT consists of one 66-kDa polypeptide chain (p66) consisting of a polymerase domain and an RNase H domain and one 51-kDa polypeptide chain (p51) containing only the polymerase domain. These two subunits interact asymmetrically to generate only one polymerase cleft that binds one primer-template, one dNTP, one noncompetitive inhibitor, and one tRNA (2-5). The polymerase domains of p51 and p66 differ by having an alternative arrangement of four subdomains (1). The three subdomains that form the large polymerase active-site cleft in p66 are called "fingers," "thumb," and "palm" by analogy ofthis polymerase structure to that ofa right hand. The fourth subdomain is called "connection" because it lies between the polymerase and RNase H active sites in p66. Although the heterodimer is the most stable dimer, with an equilibrium dissociation constant (Kd) of =1 x 10-9 M (6, 7), both p66 and p51 homodimers have been observed in vitro but are much less tightly associated (7). The major question addressed here is how a single amino acid sequence can form two quite different structures and result in such an asymmetric subunit interaction. Furthermore, and in the light of the structural observations, it is of interest to consider the likely conformation of pSi or p66 monomers as well as the possible structures of homodimers of these subunits.The crystal structure of the HIV-1 RT heterodimer complexed with a noncompetitive inhibitor, Nevirapine, was initially derived from a 3.5-A resolution electron density map (1) and has now been partially refined at 2.9-A resolution (8, 9). The structure of HIV-1 RT complexed with the Fab portion of a monoclonal antibody and duplex DNA determined at 3-A resolution shows the same structure for the RT and provides experimental evidence for the primer-template location (10). RESULTS AND DISCUSSIONThe Asymmetric Dimer Structure. The four polymerase subdomains of HIV-1 RT have very different relative orientations in the two subunits ofthe RT heterodimer (Fig. 1). The p51 subunit has a compact structure that we can refer to as "closed," while the p66 subunit has a more extended structure and a large cleft that can be referred to as "open." With the connection domains oriented identically, the different sets of interactions made by the fingers, palm, and thumb subdomains of each ofthe two subunits are clearly seen (Fig. 1). Changes in the contacts between the connection and the fingers subdomains are more modest.Interactions between the two subunits are completely asymmetric in that the subunit interface on p51 involves different amino acid residues than the...

We report the crystal structure of an NH2-terminal 388-residue fragment of T4 DNA polymerase (protein N388) refined at 2.2 A resolution. This fragment contains both the 3'-5' exonuclease active site and part of the autologous mRNA binding site (J. D. Karam, personal communication). The structure of a complex between the apoprotein N388 and a substrate, p(dT)3, has been refined at 2.5 A resolution to a crystallographic R-factor of 18.7%. Two divalent metal ion cofactors, Zn(II) and Mn(II), have been located in crystals of protein N388 which had been soaked in solutions containing Zn(II), Mn(II), or both. The structure of the 3'-5' exonuclease domain of protein N388 closely resembles the corresponding region in the Klenow fragment despite minimal sequence identity. The side chains of four carboxylate residues that serve as ligands for the two metal ions required for catalysis are located in geometrically equivalent positions in both proteins with a rms deviation of 0.87 A. There are two main differences between the 3'-5' exonuclease active site regions of the two proteins: (I) the OH of Tyr-497 in the Klenow fragment interacts with the scissile phosphate in the active site whereas the OH of the equivalent tyrosine (Tyr-320) in protein N388 points away from the active center; (II) different residues form of the binding pocket for the 3'-terminal bases of the substrate. In the protein N388 complex the 3'-terminal base of p(dT)3 is rotated approximately 60 degrees relative to the position that the corresponding base occupies in the p(dT)3 complex with the Klenow fragment. Finally, a separate domain (residues 1-96) of protein N388 may be involved in mRNA binding that results in translational regulation of T4 DNA polymerase (Pavlov & Karam, 1994).

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Site specific mutants in the pol active center of RB69 DNA polymerase have been produced and studied using rapid chemical-quench techniques. Pre-steady-state kinetic analysis carried out with Mg(2+) and Mn(2+) has enabled us to divide the mutants into two groups. One group had greatly reduced k(pols) values in the presence of Mg(2+) but responded to Mn(2+) which restored the k(pol) values for the nucleotidyl transfer reaction to near wild-type levels. The other group of mutants also had lower k(pol) values, relative to that of the wild-type polymerase, but could not be rescued by Mn(2+). The behavior of these mutants was interpreted in terms of the crystal structures of the available RB69 pol complexes. Our results on the metal ion dependence of the D621A and E686A mutants, together with knowledge of the position of their side chains in two different RB69 pol conformations, suggest that these acidic residues serve as alternative ligands for the metal ions destined to occupy the A and B catalytic sites. We infer that this occurs prior to the conformational change that produces the ternary RB69 pol complex in which the A and B metal ions are ligated by D623 and D411 as the enzyme is poised for phosphoryl transfer.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a national public health concern in the United States because of its increased transmissibility. Over 500 COVID-19 cases associated with this variant have been detected since December 2020, but its local establishment and pathways of spread are relatively unknown. Using travel, genomic, and diagnostic testing data, we highlight the primary ports of entry for B.1.1.7 in the US and locations of possible underreporting of B.1.1.7 cases. New York, which receives the most international travel from the UK, is likely one of the key hubs for introductions and domestic spread. Finally, we provide evidence for increased community transmission in several states. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

DNA polymerases from the A and B families with 3'-5' exonucleolytic activity have exonuclease domains with similar three-dimensional structures that require two divalent metal ions for catalysis. B family DNA polymerases that are part of a replicase generally have a more potent 3'-5' exonuclease (exo) activity than A family DNA polymerases that mainly function in DNA repair. To investigate the basis for these differences, we determined pH-activity profiles for the exonuclease reactions of T4, RB69, and phi29 DNA polymerases as representatives of B family replicative DNA polymerases and the Klenow fragment (KF) as an example of a repair DNA polymerase in the A family. We performed exo assays under single-turnover conditions and found that excision rates exhibited by the B family DNA polymerases were essentially independent of pH between pH 6.5 and 8.5, whereas the exo activity of KF increased 10-fold for each unit increase in pH. Three exo domain mutants of RB69 polymerase had much lower exo activities than the wild-type enzyme and exhibited pH-activity profiles similar to that of KF. On the basis of pH versus activity data and elemental effects obtained using short double-stranded DNA substrates terminating in phosphorothioate linkages, we suggest that the rate of the chemical step is reduced to the point where it becomes limiting with RB69 pol mutants K302A, Y323F, and E116A, in contrast to the wild-type enzyme where chemistry is faster than the rate-determining step that precedes it.