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

Lesbats P, MĂ©tifiot 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.

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Effet of cations on the kinetic of reactivation of dissociated INZn. One picomole of dissociated INzn 0.125 µM was preincubated with specific 21 bp ODN for 0–24 h in presence of Mg++ or Mn++ (7.5 mM) and then tested for concerted integration. Enzyme activity was compared to that of the dissociated INZn incubated under the same condition but without ODN. The percentage of activation was reported.
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Figure 8: Effet of cations on the kinetic of reactivation of dissociated INZn. One picomole of dissociated INzn 0.125 µM was preincubated with specific 21 bp ODN for 0–24 h in presence of Mg++ or Mn++ (7.5 mM) and then tested for concerted integration. Enzyme activity was compared to that of the dissociated INZn incubated under the same condition but without ODN. The percentage of activation was reported.

Mentions: In order to obtain more information on the mechanism of reactivation, we examined the kinetic of reactivation of the dissociated INZn enzyme in presence of Mg++ or Mn++. As shown in Figure 8, the kinetic of reactivation was found different when comparing both conditions. Indeed, the activation detected in presence of Mg++ was observed after 2 h and was maximal at 6 h. In contrast, when Mn++ was used the activation was not detected before 10 h. These results are consistent with the half-life of the INÄ‹DNA complexes determined before in the presence of both cations (31). Taken together these results indicate that monomer reactivation was mainly due to IN reassociation on short ODNs followed by the release of active oligomeric complexes. But the previous demonstration of the displacement of viral DNA termini from stable IN nucleoprotein complexes induced by the secondary DNA binding interaction (31) meant that we could not rule out a possible tertiary complex between IN, the short ODN and the concerted integration substrate as a possible mechanism of activation.Figure 8.


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

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

Effet of cations on the kinetic of reactivation of dissociated INZn. One picomole of dissociated INzn 0.125 µM was preincubated with specific 21 bp ODN for 0–24 h in presence of Mg++ or Mn++ (7.5 mM) and then tested for concerted integration. Enzyme activity was compared to that of the dissociated INZn incubated under the same condition but without ODN. The percentage of activation was reported.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Effet of cations on the kinetic of reactivation of dissociated INZn. One picomole of dissociated INzn 0.125 µM was preincubated with specific 21 bp ODN for 0–24 h in presence of Mg++ or Mn++ (7.5 mM) and then tested for concerted integration. Enzyme activity was compared to that of the dissociated INZn incubated under the same condition but without ODN. The percentage of activation was reported.
Mentions: In order to obtain more information on the mechanism of reactivation, we examined the kinetic of reactivation of the dissociated INZn enzyme in presence of Mg++ or Mn++. As shown in Figure 8, the kinetic of reactivation was found different when comparing both conditions. Indeed, the activation detected in presence of Mg++ was observed after 2 h and was maximal at 6 h. In contrast, when Mn++ was used the activation was not detected before 10 h. These results are consistent with the half-life of the INÄ‹DNA complexes determined before in the presence of both cations (31). Taken together these results indicate that monomer reactivation was mainly due to IN reassociation on short ODNs followed by the release of active oligomeric complexes. But the previous demonstration of the displacement of viral DNA termini from stable IN nucleoprotein complexes induced by the secondary DNA binding interaction (31) meant that we could not rule out a possible tertiary complex between IN, the short ODN and the concerted integration substrate as a possible mechanism of activation.Figure 8.

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