HIV-1 Integrase Preassembled on Donor DNA Is Refractory to Activity Stimulation by LEDGF/p75

详细信息    查看全文

• 作者：Fang Yu ; Gregg S. Jones ; Magdeleine Hung ; Alice H. Wagner ; Holly L. MacArthur ; Xiaohong Liu ; Stephanie Leavitt ; Martin J. McDermott ; Manuel Tsiang
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：March 13, 2007
• 年：2007
• 卷：46
• 期：10
• 页码：2899 - 2908
• 全文大小：228K
• 年卷期：v.46,no.10(March 13, 2007)
• ISSN：1520-4995

文摘

LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlyingmechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-wellplate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed.Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, butdisplayed 3- to 7-fold higher IC₅₀ values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. Inaddition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNAduring the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant ofcatalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step.In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in eitherthe concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysiswas observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at thestart of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. Wepropose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereasdonor DNA can engage either free or LEDGF/p75-bound integrase.