Coloma Javier, Johnson Robert E, Prakash Louise, Prakash Satya, Aggarwal Aneel K
Department of Structural &Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA.
Department of Biochemistry and Molecular Biology, 301 University Boulevard, University of Texas Medical Branch, Galveston, TX 77755-1061, USA.
Sci Rep. 2016 Apr 1;6:23784. doi: 10.1038/srep23784.
The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer--with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes.
在真核生物中，DNA聚合酶α/引发酶复合体组装短的RNA-DNA片段，用于引发滞后链和前导链的DNA复制。因此，DNA聚合酶α亚基在引物合成过程中会遇到两种类型的底物：RNA:DNA螺旋和DNA:DNA螺旋。有研究表明，聚合酶亚基与DNA:DNA螺旋的结合是真核生物中引物终止的基础。然而，关于DNA聚合酶α亚基在引物合成过程中如何实际与DNA:DNA螺旋结合，目前尚无结构信息。我们在此展示了人DNA聚合酶α亚基与DNA:DNA螺旋复合体的首个晶体结构。出乎意料的是，我们发现与聚合酶接触的DNA:DNA螺旋部分并非B型，而是A-B混合型。先前观察到的与RNA引物的几乎所有接触在与DNA引物结合时都得以保留——相同的一组聚合酶残基追踪DNA或RNA引物的糖磷酸骨架。因此，与特定接触的丧失不同，将DNA从B型扭曲为A-B混合型的自由能成本可能预示着真核生物中引物合成的终止。
