Limits...
In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state.

Lesbats P, Métifiot M, Calmels C, Baranova S, Nevinsky G, Andreola ML, Parissi V - Nucleic Acids Res. (2008)

Bottom Line: In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN.Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends.Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire MCMP, UMR 5234-CNRS, Université Victor Segalen Bordeaux 2, Bordeaux, France.

ABSTRACT
HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

Show MeSH

Related in: MedlinePlus

Model of the in vitro IN positioning on viral ends. Interaction of monomers with short nonspecific nonviral DNA induces the formation of dimers inactive for processing, strand transfer and integration activities (type 2 dimers, way A). The interaction of monomers with the specific 21 nt viral DNA ends allow the formation dimers able to catalyze the processing and strand transfer reactions (type 1 dimers, way B). In the presence of longer DNA mimicking the viral genome and containing both short specific DNA regions and larger unspecific domains and in the absence of targeting, IN (standard in vitro conditions) will bind more frequently the nonspecific sequences (way A1). This will lead to the major formation of inactive type 2 dimers (A2). In solution and especially in associated IN preparations (concentrated one or enzyme purified in absence of detergent, in presence of Zn++), preformed dimers (including types 1 and 2) are present. Type 1 dimers can bind the viral ends (B1) allowing the formation of the active tetrameric synaptic complex (SSC, B2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602759&req=5

Figure 10: Model of the in vitro IN positioning on viral ends. Interaction of monomers with short nonspecific nonviral DNA induces the formation of dimers inactive for processing, strand transfer and integration activities (type 2 dimers, way A). The interaction of monomers with the specific 21 nt viral DNA ends allow the formation dimers able to catalyze the processing and strand transfer reactions (type 1 dimers, way B). In the presence of longer DNA mimicking the viral genome and containing both short specific DNA regions and larger unspecific domains and in the absence of targeting, IN (standard in vitro conditions) will bind more frequently the nonspecific sequences (way A1). This will lead to the major formation of inactive type 2 dimers (A2). In solution and especially in associated IN preparations (concentrated one or enzyme purified in absence of detergent, in presence of Zn++), preformed dimers (including types 1 and 2) are present. Type 1 dimers can bind the viral ends (B1) allowing the formation of the active tetrameric synaptic complex (SSC, B2).

Mentions: Our in vitro data led us to propose a model described in Figure 10 for the initial attachment of IN on viral DNA. IN can bind DNA under all the different oligomeric forms including monomers. Interaction between IN monomers and DNA might induce oligomerization of the enzyme, leading to dimers and tetramers whose structures and functions depend of the bound DNA sequence. Interaction between monomers and nonspecific DNA induce the formation of dimers inactive for processing, strand transfer and integration activities (type 2) but displaying a non-sequence-specific double strand endonuclease activity (way A). The interaction of monomers with the specific 21 nt viral DNA end sequence would allow the formation of type 1 dimers able to catalyze processing and strand transfer reactions in the so-called Strand Transfer Complex (way B), (13).Figure 10.


In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state.

Lesbats P, Métifiot M, Calmels C, Baranova S, Nevinsky G, Andreola ML, Parissi V - Nucleic Acids Res. (2008)

Model of the in vitro IN positioning on viral ends. Interaction of monomers with short nonspecific nonviral DNA induces the formation of dimers inactive for processing, strand transfer and integration activities (type 2 dimers, way A). The interaction of monomers with the specific 21 nt viral DNA ends allow the formation dimers able to catalyze the processing and strand transfer reactions (type 1 dimers, way B). In the presence of longer DNA mimicking the viral genome and containing both short specific DNA regions and larger unspecific domains and in the absence of targeting, IN (standard in vitro conditions) will bind more frequently the nonspecific sequences (way A1). This will lead to the major formation of inactive type 2 dimers (A2). In solution and especially in associated IN preparations (concentrated one or enzyme purified in absence of detergent, in presence of Zn++), preformed dimers (including types 1 and 2) are present. Type 1 dimers can bind the viral ends (B1) allowing the formation of the active tetrameric synaptic complex (SSC, B2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602759&req=5

Figure 10: Model of the in vitro IN positioning on viral ends. Interaction of monomers with short nonspecific nonviral DNA induces the formation of dimers inactive for processing, strand transfer and integration activities (type 2 dimers, way A). The interaction of monomers with the specific 21 nt viral DNA ends allow the formation dimers able to catalyze the processing and strand transfer reactions (type 1 dimers, way B). In the presence of longer DNA mimicking the viral genome and containing both short specific DNA regions and larger unspecific domains and in the absence of targeting, IN (standard in vitro conditions) will bind more frequently the nonspecific sequences (way A1). This will lead to the major formation of inactive type 2 dimers (A2). In solution and especially in associated IN preparations (concentrated one or enzyme purified in absence of detergent, in presence of Zn++), preformed dimers (including types 1 and 2) are present. Type 1 dimers can bind the viral ends (B1) allowing the formation of the active tetrameric synaptic complex (SSC, B2).
Mentions: Our in vitro data led us to propose a model described in Figure 10 for the initial attachment of IN on viral DNA. IN can bind DNA under all the different oligomeric forms including monomers. Interaction between IN monomers and DNA might induce oligomerization of the enzyme, leading to dimers and tetramers whose structures and functions depend of the bound DNA sequence. Interaction between monomers and nonspecific DNA induce the formation of dimers inactive for processing, strand transfer and integration activities (type 2) but displaying a non-sequence-specific double strand endonuclease activity (way A). The interaction of monomers with the specific 21 nt viral DNA end sequence would allow the formation of type 1 dimers able to catalyze processing and strand transfer reactions in the so-called Strand Transfer Complex (way B), (13).Figure 10.

Bottom Line: In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN.Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends.Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire MCMP, UMR 5234-CNRS, Université Victor Segalen Bordeaux 2, Bordeaux, France.

ABSTRACT
HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

Show MeSH
Related in: MedlinePlus