Fan L, Kaguni L S
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA.
Biochemistry. 2001 Apr 17;40(15):4780-91. doi: 10.1021/bi010102h.
Drosophila mitochondrial DNA polymerase, pol gamma, is a heterodimeric complex of catalytic subunit and accessory subunits. Physical interactions between the two subunits were investigated by deletion mutagenesis in both in vivo reconstitution and in vitro protein overlay analyses. Our results suggest that the accessory subunit may consist of three domains, designated the N, M, and C domains. The M and C regions comprise the major contacts involved in subunit interaction, likely with multiple sites in the exonuclease (exo) region and part of the spacer between the exo and DNA polymerase (pol) regions in the catalytic subunit. Furthermore, the N region in the accessory subunit may modulate subunit assembly and/or conformation through weak interaction with the pol region in the catalytic subunit. Sequence comparisons identify a significant similarity between the M region of the accessory subunit and the RNase H domain of HIV-1 reverse transcriptase. On the basis of these results, the proposed function of the C-terminus of the accessory subunit in RNA primer recognition, and previous observations that mitochondrial DNA polymerase is itself a reverse transcriptase, we propose that the overall conformation and arrangement of functional regions in the Drosophila pol gamma complex resemble those of HIV-1 reverse transcriptase.
果蝇线粒体DNA聚合酶γ是一种由催化亚基和辅助亚基组成的异源二聚体复合物。通过体内重组中的缺失诱变和体外蛋白质覆盖分析，研究了两个亚基之间的物理相互作用。我们的结果表明，辅助亚基可能由三个结构域组成，分别命名为N、M和C结构域。M和C区域构成了亚基相互作用中涉及的主要接触部位，可能与催化亚基中外切核酸酶（exo）区域的多个位点以及exo和DNA聚合酶（pol）区域之间的间隔区的一部分相互作用。此外，辅助亚基中的N区域可能通过与催化亚基中的pol区域的弱相互作用来调节亚基组装和/或构象。序列比较发现辅助亚基的M区域与HIV-1逆转录酶的RNase H结构域之间存在显著相似性。基于这些结果、辅助亚基C末端在RNA引物识别中的拟议功能以及先前关于线粒体DNA聚合酶本身是逆转录酶的观察结果，我们提出果蝇polγ复合物中功能区域的整体构象和排列类似于HIV-1逆转录酶。
