Yu Fang, Jones Gregg S, Hung Magdeleine, Wagner Alice H, MacArthur Holly L, Liu Xiaohong, Leavitt Stephanie, McDermott Martin J, Tsiang Manuel
Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, USA.
Biochemistry. 2007 Mar 13;46(10):2899-908. doi: 10.1021/bi602387u. Epub 2007 Feb 14.
LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate 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, but displayed 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. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis 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 either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was 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 the start 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. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.
已知LEDGF/p75可在体外增强整合酶链转移活性，但其潜在机制尚不清楚。采用适用于96孔板形式的化学发光读数的整合酶测定法，分析了LEDGF/p75对3'-加工和链转移步骤的影响。链转移反应的整合酶抑制剂在LEDGF/p75存在下仍保持活性，但IC50值高3至7倍。我们的分析表明，在存在150 nM LEDGF/p75的情况下，3'-加工步骤中活性整合酶/供体DNA复合物增加了5.3倍。此外，这些整合酶/供体DNA复合物在随后的链转移步骤中对靶DNA的亲和力高4.5倍。我们还观察到，当在3'-加工步骤中存在150 nM LEDGF/p75时，链转移步骤的催化速率常数增加了3.7倍。相反，当在链转移步骤开始时添加LEDGF/p75时，未观察到活性整合酶/供体DNA复合物浓度或其链转移催化速率常数增加。该观察结果表明，在不存在LEDGF/p75的情况下形成的整合酶/供体DNA对LEDGF/p75的刺激作用变得不敏感。相反，在链转移步骤开始时添加的这种LEDGF/p75能够促进新的活性整合酶/供体DNA复合物群体的形成，这些复合物在添加LEDGF/p75后45分钟延迟后开始发挥作用。我们提出了一个模型，即LEDGF/p75只能在整合酶结合供体DNA之前结合整合酶，而供体DNA可以与游离的或与LEDGF/p75结合的整合酶结合。
