dNTP Binding to HIV-1 Reverse Transcriptase and Mammalian DNA Polymerase β as Revealed by Affinity Labeling with a Photoreactive dNTP Analog

Abstract

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase ␤ (␤-pol) were labeled using a photoreactive analog of dCTP, exo-N-[␤-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5-triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP crosslinking was increased in the presence of a dideoxyterminated primer-template with RT, but not with ␤-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of ␤-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.DNA polymerases must recognize and incorporate the correct dNTP with high fidelity to maintain genetic stability. The interaction of the polymerase with the DNA primer-template is essentially nonspecific, since the enzyme must bind to an "infinite" number of sequences during replication, while dNTPtemplate base recognition must be highly specific for DNA replication of high fidelity. Since the role of the polymerase in dNTP recognition and selection is poorly understood, elucidation of polymerase-dNTP interactions under a variety of conditions will aid our understanding of this fundamental process.During the last several years our knowledge of the structure of DNA polymerases and their interactions with substrates has evolved significantly. The three-dimensional structures of several DNA polymerases have been solved by x-ray crystallography including the Klenow fragment of DNA polymerase I (1), HIV-1 1 reverse transcriptase (RT) (2-5), Taq polymerase (6), and rat/human DNA polymerase ␤ (␤-pol) (7-11). The overall architecture of these enzymes is similar, including a cleft that binds DNA. These polymerases have been described using analogy to the anatomical features of a hand as fingers, palm, and thumb subdomains (2). Catalytically essential carboxylates that bind to the incoming deoxynucleoside 5Ј-triphosphate (dNTP) via Mg 2ϩ are found in the palm subdomain. Whereas the palm subdomains of these polymerases are structurally similar, the finger and thumb subdomains are structurally distinct. The dNTP binding pocket for DNA polymerase ␤ has been defined by the x-ray structure of the ␤-pol⅐ddCTP⅐primer-template complex (9). The triphosphate moiety of the incoming ddCTP is observed to be interacting with re...

The Fidelity of DNA Polymerase β during Distributive and Processive DNA Synthesis

“…This alteration in base stacking interactions has been suggested to be relevant to the fidelity of a single-nucleotide gapfilling reaction (31). Moreover, cross-linking studies suggested that the 8-kDa domain also contributes to dNTP binding in the polymerase active site (32,33). Thus, unlike distributive synthesis on a long single-stranded template, pol ␤ fills gaps of up to 6 nucleotides in a processive manner, and the 8-kDa domain participates in this reaction in multiple ways.…”

“…A possible explanation is that the 8-kDa domain, which binds to the downstream 5Ј terminus, contributes to polymerase selectivity during synthesis of the final nucleotide in a gap. Biochemical evidence for this comes from cross-linking studies which suggest that the 8-kDa domain contributes to dNTP binding in the polymerase active site (32,33). The crystal structure of a pol ␤⅐DNA⅐ddNTP complex (30) reveals a 90°kink in the DNA at the 5Ј-phosphodiester linkage of the template residue in the polymerase active site.…”

DNA Polymerase λ, a Novel DNA Repair Enzyme in Human Cells

“…Indeed, replacing Asp 276 with an uncharged residue (i.e. valine or glycine) results in an apparent increase in the nucleotide binding affinity (37). Moreover, a D276V mutant of pol ␤ has a 4-9-fold increased nucleotide binding affinity compared with that of the wild-type enzyme (36).…”

Loss of DNA Polymerase β Stacking Interactions with Templating Purines, but Not Pyrimidines, Alters Catalytic Efficiency and Fidelity

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