Structure of HIV‐1 reverse transcriptase/d4TTP complex: Novel DNA cross‐linking site and pH‐dependent conformational changes

Stavudine (d4T, 2′,3′‐didehydro‐2′,3′‐dideoxythymidine) was one of the first chain‐terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV‐1 RT/dsDNA template‐primer. We also present a new strategy for disulfide (S–S) chemical cross‐linking between N⁶ of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross‐link site is upstream of the duplex‐binding region of RT, however, the structure is very similar to published RT structures with cross‐linking to Q258C in the thumb, which suggests that cross‐linking at either site does not appreciably perturb the RT/DNA structures. RT has a catalytic maximum at pH 7.5. We determined the X‐ray structures of the I63C‐RT/dsDNA/d4TTP cross‐linked complexes at pH 7, 7.5, 8, 8.5, 9, and 9.5. We found small (~0.5 Å), pH‐dependent motions of the fingers subdomain that folds in to form the dNTP‐binding pocket. We propose that the pH‐activity profile of RT relates to this motion of the fingers. Due to side effects of neuropathy and lipodystrophy, use of d4T has been stopped in most countries, however, chemical modification of d4T might lead to the development of a new class of nucleoside analogs targeting RNA and DNA polymerases.     

Kinetic control of Mg2+-dependent melting of duplex DNA ends by Escherichia coli RecBC

Escherichia coli RecBCD is a highly processive DNA helicase involved in double-strand break repair and recombination that possesses two helicase/translocase subunits with opposite translocation directionality (RecB (3′ to 5′) and RecD (5′ to 3′)). RecBCD has been shown to melt out ∼ 5-6 bp upon binding to a blunt-ended duplex DNA in a Mg²⁺-dependent, but ATP-independent reaction. Here, we examine the binding of E. coli RecBC helicase (minus RecD), also a processive helicase, to duplex DNA ends in the presence and in the absence of Mg²⁺ in order to determine if RecBC can also melt a duplex DNA end in the absence of ATP. Equilibrium binding of RecBC to DNA substrates with ends possessing pre-formed 3′ and/or 5′ single-stranded (ss)-(dT)_n flanking regions (tails) (n ranging from zero to 20 nt) was examined by competition with a fluorescently labeled reference DNA and by isothermal titration calorimetry. The presence of Mg²⁺ enhances the affinity of RecBC for DNA ends possessing 3′ or 5′-(dT)_n ssDNA tails with n < 6 nt, with the relative enhancement decreasing as n increases from zero to six nt. No effect of Mg²⁺ was observed for either the binding constant or the enthalpy of binding (ΔH_obs) for RecBC binding to DNA with ssDNA tail lengths, n ≥ 6 nucleotides. Upon RecBC binding to a blunt duplex DNA end in the presence of Mg²⁺, at least 4 bp at the duplex end become accessible to KMnO₄ attack, consistent with melting of the duplex end. Since Mg²⁺ has no effect on the affinity or binding enthalpy of RecBC for a DNA end that is fully pre-melted, this suggests that the role of Mg²⁺ is to overcome a kinetic barrier to melting of the DNA by RecBC and presumably also by RecBCD. These data also provide an accurate estimate (ΔH_obs = 8 ± 1 kcal/mol) for the average enthalpy change associated with the melting of a DNA base-pair by RecBC.     

Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit

Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wild-type gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA—involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds double-stranded DNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143-152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143-152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation.     

The Escherichia coli PriA Helicase-Double-Stranded DNA Complex: Location of the Strong DNA-Binding Subsite on the Helicase Domain of the Protein and the Affinity Control by the Two Nucleotide-Binding Sites of the Enzyme

The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA-dsDNA complex is only 5 ± 1 bp, that is, dramatically lower than 20 ± 3 nucleotides occluded in the enzyme-single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA protein. The dsDNA intrinsic affinity is considerably higher than the ssDNA affinity and the binding process is accompanied by a significant positive cooperativity. Association of cofactors with strong and weak nucleotide-binding sites of the protein profoundly affects the intrinsic affinity and the cooperativity, without affecting the stoichiometry. ATP analog binding to either site diminishes the intrinsic affinity but preserves the cooperativity. ADP binding to the strong site leads to a dramatic increase of the cooperativity and only slightly affects the affinity, while saturation of both sites with ADP strongly increases the affinity and eliminates the cooperativity. Thus, the coordinated action of both nucleotide-binding sites on the PriA-dsDNA interactions depends on the structure of the phosphate group. The significance of these results for the enzyme activities in recognizing primosome assembly sites or the ssDNA gaps is discussed.     

Sin Resolvase Catalytic Activity and Oligomerization State are Tightly Coupled

Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3′-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex.