Morris P D, Tackett A J, Babb K, Nanduri B, Chick C, Scott J, Raney K D
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
J Biol Chem. 2001 Jun 8;276(23):19691-8. doi: 10.1074/jbc.M010928200. Epub 2001 Feb 27.
The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species. The specific activity for ssDNA stimulated ATPase activity was independent of Dda concentration. Dda was mutated to produce an ATPase-deficient protein (K38A Dda) by altering a residue within a conserved, nucleotide binding loop. The helicase activity of K38A Dda was inactivated, although DNA binding properties were similar to Dda. In the presence of limiting DNA substrate, the rate of unwinding by Dda was not changed; however, the amplitude of product formation was reduced in the presence of increasing concentrations of K38A Dda. The reduction was between that expected for a monomeric or dimeric helicase based on simple competition for substrate binding. When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no reduction in the observed rate for strand separation. Taken together, these results indicate that oligomerization of Dda is not required for DNA unwinding.
许多解旋酶的活性形式是寡聚体，这可能是因为寡聚化提供了DNA解旋所需的多个DNA结合位点。为了了解噬菌体T4 Dda解旋酶的机制，研究了寡聚化的潜在需求。化学交联和高压凝胶过滤色谱几乎没有提供形成寡聚体的证据。单链DNA刺激的ATP酶活性的比活性与Dda浓度无关。通过改变保守的核苷酸结合环内的一个残基，将Dda突变为产生一种ATP酶缺陷蛋白（K38A Dda）。K38A Dda的解旋酶活性失活，尽管其DNA结合特性与Dda相似。在有限的DNA底物存在下，Dda的解旋速率没有改变；然而，在K38A Dda浓度增加的情况下，产物形成的幅度降低。这种降低介于基于简单底物结合竞争的单体或二聚体解旋酶预期的降低之间。当在过量DNA底物存在下测量DNA解旋时，添加K38A Dda不会导致观察到的链分离速率降低。综上所述，这些结果表明Dda的寡聚化对于DNA解旋不是必需的。
