Reems J A, Wood S, McHenry C S
Department of Biochemistry, University of Colorado Health Sciences Center, Denver 80262.
J Biol Chem. 1995 Mar 10;270(10):5606-13. doi: 10.1074/jbc.270.10.5606.
Escherichia coli DNA polymerase III holoenzyme forms a stable initiation complex with RNA-primed template in the presence of ATP. To determine the linear arrangement of the holoenzyme subunits along the primer-template duplex region, we cross-linked holoenzyme to a series of photo-reactive primers. Site-specific photo-cross-linking revealed that the alpha, beta, and gamma subunits formed ATP-dependent contacts with the primer-template. The alpha-polymerase catalytic subunit covalently attached to nucleotide positions -3, -9, and -13 upstream of the primer terminus, with the most efficient adduct formation occurring at position -9. The gamma subunit contacted the primer at positions -13, -18, and -22, with the strongest gamma-primer interactions occurring at position -18. The beta subunit predominated in cross-linking at position -22. Thus, within the initiation complex, alpha contacts roughly the first 13 nucleotides upstream of the 3'-primer terminus followed by gamma at -18 and beta at -22, and the gamma subunit remains a part of the initiation complex, bridging the alpha and beta subunits. Analyses of the interaction of photo-activatible primer-templates with the preinitiation complex proteins (gamma-complex (gamma-delta-delta'-chi-psi) and beta subunit) revealed the gamma subunit within the preinitiation complex covalently attached to primer at position -3. However, addition of core DNA polymerase III to preinitiation complex, fully reconstituting holoenzyme resulted in replacement of gamma by alpha at the primer terminus. These data indicate that assembly of holoenzyme onto a primer-template can occur in distinct stages and results in a structural rearrangement during initiation complex formation.
在ATP存在的情况下，大肠杆菌DNA聚合酶III全酶与RNA引发的模板形成稳定的起始复合物。为了确定全酶亚基沿引物-模板双链区域的线性排列，我们将全酶与一系列光反应性引物进行交联。位点特异性光交联显示，α、β和γ亚基与引物-模板形成了ATP依赖性接触。α聚合酶催化亚基与引物末端上游的核苷酸位置-3、-9和-13共价连接，最有效的加合物形成发生在位置-9。γ亚基在位置-13、-18和-22与引物接触，γ与引物之间最强的相互作用发生在位置-18。β亚基在位置-22的交联中占主导。因此，在起始复合物中，α大致接触3'-引物末端上游的前13个核苷酸，随后是-18位置的γ和-22位置的β，并且γ亚基仍然是起始复合物的一部分，连接α和β亚基。对光活化引物-模板与起始前复合物蛋白（γ复合物（γ-δ-δ'-χ-ψ）和β亚基）相互作用的分析表明，起始前复合物中的γ亚基在位置-3与引物共价连接。然而，将核心DNA聚合酶III添加到起始前复合物中，完全重组全酶会导致引物末端的γ被α取代。这些数据表明全酶组装到引物-模板上可以分不同阶段进行，并在起始复合物形成过程中导致结构重排。
