Rapid kinetics of protein-nucleic acid interaction is a major component of HIV-1 nucleocapsid protein's nucleic acid chaperone function
Rapid kinetics of protein-nucleic acid interaction is a major component of HIV-1 nucleocapsid protein's nucleic acid chaperone function
复制标题
DOI：
10.1016/j.jmb.2006.08.070
复制
发表时间：
2006-11-10
影响因子：
5.6
通讯作者：
Williams, Mark C.
中科院分区：
文献类型：
--
作者：
Cruceanu, Margareta;Gorelick, Robert J.;Williams, Mark C.
人类免疫缺陷病毒1型（HIV-1）核衣壳蛋白（NC）的核酸伴侣活性在逆转录病毒生命周期中起着重要作用，部分是通过促进整个逆转录过程中的许多核酸重排。最近的研究已经确定双链体不稳定和核酸聚集作为NC的伴侣活性的两个主要组成部分。为了更好地理解NC的功能结构域对这两种活性的贡献，我们使用光镊在野生型或突变型HIV-1 NC存在下通过螺旋-卷曲转变来拉伸单个λ DNA分子。蛋白质诱导的双链体不稳定直接测量为力诱导的熔融自由能的平均降低，而NC促进链退火的能力由DNA拉伸-松弛循环中的滞后量确定。通过研究全长和截短NC的无锌变体，阐明了NC的锌指和N-末端碱性结构域对分子伴侣活性的两个主要组分的相对贡献。此外，对含有影响一个或两个锌指基序的突变的NC变体的检查表明，有效的链退火活性与NC快速结合核酸和与核酸解离的能力相关。具有慢开/关速率的NC变体在链退火中是低效的，即使它们仍然能够进行高亲和力核酸结合、双链体去稳定化和/或核酸聚集。两者合计，这些观察结果建立了快速动力学的蛋白质-核酸相互作用的另一个主要组成部分NC的伴侣功能。(c)2006爱思唯尔有限公司保留所有权利。
The nucleic acid chaperone activity of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) plays an important role in the retroviral life cycle, in part, by facilitating numerous nucleic acid rearrangements throughout the reverse transcription process. Recent studies have identified duplex destabilization and nucleic acid aggregation as the two major components of NC's chaperone activity. In order to better understand the contribution of the functional domains of NC to these two activities, we used optical tweezers to stretch single lambda DNA molecules through the helix-coil transition in the presence of wild-type or mutant HIV-1 NC. Protein-induced duplex destabilization was measured directly as an average decrease of the force-induced melting free energy, while NC's ability to facilitate strand annealing was determined by the amount of hysteresis in the DNA stretch-relax cycle. By studying zinc-free variants of full-length and truncated NC, the relative contributions of NC's zinc fingers and N-terminal basic domain to the two major components of chaperone activity were elucidated. In addition, examination of NC variants containing mutations affecting one or both zinc finger motifs showed that effective strand annealing activity is correlated with NC's ability to rapidly bind and dissociate from nucleic acids. NC variants with slow on/off rates are inefficient in strand annealing, even though they may still be capable of high affinity nucleic acid binding, duplex destabilization, and/or nucleic acid aggregation. Taken together, these observations establish the rapid kinetics of protein-nucleic acid interaction as another major component of NC's chaperone function. (c) 2006 Elsevier Ltd. All rights reserved.