A novel structure of DNA repair protein RecO from Deinococcus radiodurans

Recovery of arrested replication requires coordinated action of DNA repair, replication, and recombination machineries. Bacterial RecO protein is a member of RecF recombination repair pathway important for replication recovery. RecO possesses two distinct activities in vitro, closely resembling those of eukaryotic protein Rad52: DNA annealing and RecA-mediated DNA recombination. Here we present the crystal structure of the RecO protein from the extremely radiation resistant bacteria Deinococcus radiodurans (DrRecO) and characterize its DNA binding and strand annealing properties. The RecO structure is totally different from the Rad52 structure. DrRecO is comprised of three structural domains: an N-terminal domain which adopts an OB-fold, a novel alpha-helical domain, and an unusual zinc-binding domain. Sequence alignments suggest that the multidomain architecture is conserved between RecO proteins from other bacterial species and is suitable to elucidate sites of protein-protein and DNA-protein interactions necessary for RecO functions during the replication recovery and DNA repair.     

Substituted hydrophobic and hydrophilic residues at methionine-68 influence the chaperone-like function of αB-crystallin

Amino acid residues 57–69 in B-crystallin have been implicated as a target protein binding site. Moreover, a direct correlation between the extent of -crystallin hydrophobicity and chaperone-like activity has been demonstrated. The purpose of this study was to mutate a moderately hydrophobic residue Met-68 (M-68) in the above region to strongly hydrophobic and hydrophilic residues and show whether chaperoning ability is affected with or without structural changes. Mutation of M-68 to Val, Ile or Thr did not result in significant changes in molecular mass and secondary and tertiary structures. However, the Val and Ile mutants showed significant improvement and the Thr mutant showed substantial loss in chaperone activity. Differences in chaperone function in the absence of any structural changes confirmed that the hydrophobicity or hydrophilicity of the substituted amino acid in the putative target protein binding site was the only contributing factor.     

Molecular Interactions between HIV-1 Integrase and the Two Viral DNA Ends within the Synaptic Complex that Mediates Concerted Integration

A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analysis is difficult. We investigated the architecture of HIV-1 nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway, and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3′ OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated by native agarose gel electrophoresis. In-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5′-end labeled viral DNA ends in the synaptic complex (0.68 ± 0.09) was significantly different from that observed in the strand transfer complex (0.07 ± 0.02). The calculated distances were 46 ± 3 Å and 83 ± 5 Å, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to ∼ 32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5, suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger multimer (> 120 kDa). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends before the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.     

A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity

An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported. Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size. The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys. To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells. The wild-type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding. Mutation of Arg-116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes. The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure. Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin.     

HIV-1 integrase strand transfer inhibitors stabilize an integrase-single blunt-ended DNA complex

Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC₅₀ values of ∼ 20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of > 200 nM STI, termed the IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼ 32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼ 20-25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤ 5% of input DNA). The formation of the ISD complex was not dependent on 3′OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼ 25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼ 3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex.     

A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding

Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 3′ DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves as a viral DNA binding surface for RSV IN.     

The HIV-1 integrase monomer induces a specific interaction with LTR DNA for concerted integration

The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO(4) was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3'-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19-42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.     

Rous Sarcoma Virus Synaptic Complex Capable of Concerted Integration Is Kinetically Trapped by Human Immunodeficiency Virus Integrase Strand Transfer Inhibitors

We determined conditions to produce milligram quantities of the soluble Rous sarcoma virus (RSV) synaptic complex that is kinetically trapped by HIV strand transfer inhibitors (STIs). Concerted integration catalyzed by RSV integrase (IN) is effectively inhibited by HIV STIs. Optimized assembly of the RSV synaptic complex required IN, a gain-of-function 3′-OH-recessed U3 oligonucleotide, and an STI under specific conditions to maintain solubility of the trapped synaptic complex at 4 °C. A C-terminal truncated IN (1–269 residues) produced a homogeneous population of trapped synaptic complex that eluted at ∼151,000 Da upon Superdex 200 size-exclusion chromatography (SEC). Approximately 90% of input IN and DNA are incorporated into the trapped synaptic complex using either the C-terminally truncated IN or wild type IN (1–286 residues). No STI is present in the SEC running buffer suggesting the STI-trapped synaptic complex is kinetically stabilized. The yield of the trapped synaptic complex correlates with the dissociative half-life of the STI observed with HIV IN-DNA complexes. Dolutegravir, MK-2048, and MK-0536 are equally effective, whereas raltegravir is ∼70% as effective. Without an STI present in the assembly mixture, no trapped synaptic complex was observed. Fluorescence and mass spectroscopy analyses demonstrated that the STI remains associated with the trapped complex. SEC-multiangle light scattering analyses demonstrated that wild type IN and the C-terminal IN truncation are dimers that acted as precursors to the tetramer. The purified STI-trapped synaptic complex contained a tetramer as shown by cross-linking studies. Structural studies of this three-domain RSV IN in complex with viral DNA may be feasible.     

Structural organization of avian retrovirus integrase in assembled intasomes mediating full-site integration

Retrovirus preintegration complexes (PIC) purified from virus-infected cells are competent for efficient concerted integration of the linear viral DNA ends by integrase (IN) into target DNA (full-site integration). In this report, we have shown that the assembled complexes (intasomes) formed in vitro with linear 3.6-kbp DNA donors possessing 3'-OH-recessed attachment (att) site sequences and avian myeloblastosis virus IN (4 nm) were as competent for full-site integration as isolated retrovirus PIC. The att sites on DNA with 3'-OH-recessed ends were protected by IN in assembled intasomes from DNase I digestion up to approximately 20 bp from the terminus. Several DNA donors containing either normal blunt-ended att sites or different end mutations did not allow assembly of complexes that exhibit the approximately 20-bp DNase I footprint at 14 degrees C. At 50 and 100 mm NaCl, the approximately 20-bp DNase I footprints were produced with wild type (wt) U3 and gain-of-function att site donors for full-site integration as previously observed at 320 mm NaCl. Although the wt U5 att site donors were fully competent for full-site integration at 37 degrees C, the approximately 20-bp DNase I footprint was not observed under a variety of assembly conditions including low NaCl concentrations at 14 degrees C. Under suboptimal assembly conditions for intasomes using U3 att DNA, DNase I probing demonstrated an enhanced cleavage site 9 bp from the end of U3 suggesting that a transient structural intasome intermediate was identified. Using a single nucleotide change at position 7 from the end and a series of small size deletions of wt U3 att site sequences, we determined that sequences upstream of the 11th nucleotide position were not required by IN to produce the approximately 20-bp DNase I footprint and full-site integration. The results suggest the structural organization of IN at the att sites in reconstituted intasomes was similar to that observed in PIC.