DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

Deprez, Eric; Tauc, Patrick; Leh, Hervé; Mouscadet, Jean‐François; Auclair, Christian; Hawkins, Mary E.; Brochon, Jean‐Claude

doi:10.1073/pnas.181024498

Cited by 83 publications

(78 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We note that the oligomerization state of other DNA-binding proteins such as repressor (54) and human immunodeficiency virus integrase (55) has been shown to be influenced by the presence of DNA. In the case of human immunodeficiency virus integrase, time-resolved anisotropy measurements of its tryptophan rotation reorientation times show that integrase exists in various oligomeric states in solution, and Mg 2ϩ and DNA cause dissociation of the tetrameric form to the monomeric form that is found in complex with DNA (55,56).…”

Section: Discussionmentioning

confidence: 99%

RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K D ‫؍‬ 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.The genes encoding the variable domains of immunoglobulins or T cell receptors are generated during lymphocyte differentiation by a somatic recombination reaction known as V(D)J recombination (1). The reaction is initiated by DNA double strand breaks created at the junction between two coding segments (termed V, D, or J) and their flanking recombination signal sequences (RSSs). 1 Cleavage is followed by a complex repair process that results in imprecise joining of the two coding segments and typically precise joining of the two RSSs. The RSSs consist of two conserved sequence elements, the heptamer (consensus 5Ј-CACAGTG-3Ј) and the nonamer (consensus 5Ј-ACAAAAACC-3Ј), separated by a poorly conserved spacer sequence of either 12 or 23 base pairs. Efficient recombination occurs only between a 12-RSS and a 23-RSS, a phenomenon known as the 12/23 rule (for review, see Ref.2). Recognition of 12/23-RSSs and concerted cleavage at the RSScoding sequence border is performed by a complex of the RAG1 and RAG2 proteins (for reviews, see Ref. 3 and 4), the lymphoid-specific products of the recombination-activating genes RAG1 and RAG2 (5, 6). Binding and cleavage of DNA by the RAG1⅐RAG2 complex is facilitated by the ubiquitously expressed architectural DNA-binding proteins 8).Deletion mutagenesis has established the minimal "core" domains of murine RAG1 (residues 384 -1008 of the 1040 aa RAG1 protein; Fig. 1A) and RAG2 (residues 1-383 of the 527 aa RAG2 protein) required for recombination activity in transfected nonlymphoid cell lines (9 -12). Most bi...

show abstract

Section: Discussionmentioning

confidence: 99%

RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Second, small peptides have been described that inhibit DNA binding to IN by shifting the IN oligomerization equilibrium from an active dimer toward an inactive tetramer (9). Third, DNA binding induces dissociation of the multimeric IN (27). Taken together, these observations suggest that a tetrameric form of the apo IN must dissociate to bind DNA and then reorganize into a tetrameric IN-DNA complex.…”

Section: Dna Tethering Overcomes In Requirement Of the Ntd For Strandmentioning

confidence: 99%

Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

Alian¹,

Griner²,

Chiang³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

IN consists of 3 functional domains: the N-terminal domain (NTD; residues 1-51) that contains a conserved ''HH-CC'' zincbinding motif, the catalytic core domain (CCD; residues 52-210) with the catalytic residues (D64, D116, and E152), and the Cterminal domain (CTD; residues 210-288) that contributes to DNA binding (2). In solution, recombinant IN exists in a dynamic equilibrium between monomers, dimers, tetramers, and higherorder oligomers (3, 4). Monomers are reportedly inactive in vitro, whereas dimers are able to catalyze 3Ј processing and integration of 1 viral end (4-9). Tetramers, which have also been isolated from human cells expressing HIV-1 IN (10), can catalyze integration of 2 viral DNA ends into target DNA (7, 11), but the exact nature of the IN complex mediating 3Ј processing and strand transfer reactions remains to be determined. The integration step is an attractive drug target given its essential role in the viral life cycle and the lack of a cellular IN homologue. Strand transfer inhibitors appear to bind significantly better to IN when it is assembled on its DNA substrate than to IN alone (12). To date there is only 1 structure of an inhibitor bound to IN (13), and that is in the absence of DNA. The compound binds at the active site; however, it dimerizes across a crystallographic 2-fold axis and therefore might not be in its bioactive configuration.Structure-based understanding of the mechanisms of the action of IN inhibitors and optimization of compounds as potential drugs targeting HIV-1 IN have been hampered by the inability to capture and crystallize IN-DNA complexes. Two key factors have contributed to this problem: first, the high salt concentration (Ϸ1 M NaCl) required to maintain full-length IN in solution interferes with DNA binding; second, IN has intrinsically low affinity for DNA. To overcome these 2 obstacles, we used disulfide cross-linking to generate soluble, catalytically-active, covalent IN-DNA complexes. A similar strategy, covalent disulfide cross-linking between HIV-1 reverse transcriptase (RT) and DNA, mediated crystallization of the .Previous cross-linking from cysteinal mutations in the CTD (6) and CCD (15) Here, we describe an IN cysteine mutant, IN Y143C , which is able to form IN-DNA complexes efficiently. The IN Y143C -DNA complexes form stable tetramers in solution, retain single-end strand transfer activity, show increased resistance to protease and nuclease digestion, and bind a strand transfer inhibitor. This IN-DNA complex can serve as an in vitro platform to identify and evolve strand transfer inhibitors of HIV integration and as a means of understanding the basis for a key part of the integration reaction.

show abstract

“…Fluorescence experiments were performed as previously described (10,11). Excitation and emission wavelengths were 298 and 350 nm, respectively.…”

Section: Plasmids and Vectorsmentioning

confidence: 99%

Disulfide-Linked Integrase Oligomers Involving C280 Residues Are Formed In Vitro and In Vivo but Are Not Essential for Human Immunodeficiency Virus Replication

Bischerour

Leh²,

Deprez

et al. 2003

J Virol

Self Cite

View full text Add to dashboard Cite

The insertion of a proviral DNA copy of the human immunodeficiency virus (HIV) genome into the host cell DNA is a key step of the retroviral cycle: it ensures expression and perpetuation of the viral genome (4). The process is carried out by the viral integrase (IN), encoded by the viral pol gene. IN catalyses two independent and successive reactions. The first reaction, 3Ј processing, consists of the cleavage of both 3Ј dinucleotides from each strand of the linear viral DNA, leaving overhanging CA ends. In the second reaction, strand transfer, the 3Ј-processed ends are used as nucleophilic agents to attack phosphodiester bonds on opposite strands of the target DNA, resulting in the joining of processed 3Ј ends to 5Ј phosphates in the target DNA (4). HIV IN is a 288-amino-acid protein composed of three functionally distinct domains (1 (16,18). Despite extensive studies of the oligomeric status of the enzyme in solution, the precise oligomerization state required for the various steps of the integration process remains unknown. As deduced for the consensus dimer of the core domain, the distance between the two active sites is not compatible with the 5-bp gap which separates the two integration sites (15). This suggests that the minimal functional oligomer is a tetramer. The three isolated domains as well as proteins consisting of two domains are capable of forming dimers. Higherorder oligomers are observed only in solutions of the fulllength protein (6,9,12,19,20,27). Discordant findings in vitro concerning the exact nature of the oligomeric form can be explained in part by the sensitivity of oligomerization to the purification process. For instance, IN was mainly monomeric when purified in the presence of detergent whereas dimers and tetramers were recovered in the absence of detergent (10,19). Nevertheless, recombinant IN prepared under reducing conditions has always been obtained as noncovalent oligomers. In contrast, recent reports suggested that covalent disulfidelinked oligomers of IN are present in virions (2, 23). We aimed to identify the residues responsible for the disulfide bridging in viral particles and to clarify the relationship between the formation of covalent dimers and IN activity both in vitro and in the context of the viral replication. We show that IN from a baculovirus expression system and from viral particles can form disulfide-linked oligomers. The mutation of the cysteine 280 residue is sufficient to abolish this disulfide bridging. However, the activity of the wild-type and C280S mutant are similar, and the virus infectivity in either dividing or noncycling cells was not affected by a C280S IN mutation. Thus, the disulfidebridged form of the IN oligomers found in the viral particles is not essential for viral replication.

show abstract

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

Cited by 83 publications

References 38 publications

RAG1-DNA Binding in V(D)J Recombination

RAG1-DNA Binding in V(D)J Recombination

Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

Disulfide-Linked Integrase Oligomers Involving C280 Residues Are Formed In Vitro and In Vivo but Are Not Essential for Human Immunodeficiency Virus Replication

Contact Info

Product

Resources

About