Byrd Alicia K, Raney Kevin D
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Biochemistry. 2005 Oct 4;44(39):12990-7. doi: 10.1021/bi050703z.
Dda has been shown previously to be active as a monomer for DNA unwinding [Nanduri et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14722] and streptavidin displacement [Byrd and Raney (2004) Nat. Struct. Mol. Biol. 11, 531]. However, its activity for streptavidin displacement increased as a function of the length of single-stranded DNA. We investigated whether Dda exhibited enhanced DNA unwinding of partially duplex DNA substrates as a function of increasing the length of the single-stranded overhangs. DNA substrates were prepared containing 16 base pairs and single-stranded overhangs of 4, 6, 8, 12, 16, 20, and 24 nucleotides. Under single turnover conditions in the presence of excess enzyme, the quantity of DNA unwound increased significantly as the length of the single strand overhang increased. Increased processivity was observed when the DNA substrate contained longer single-stranded overhangs. Equilibrium binding studies indicated that Dda bound to the substrates containing the longer overhangs significantly better than the shorter overhangs. To determine whether the increased processivity for unwinding was due to multiple molecules of Dda or due to the increased binding affinity to the longer overhangs, DNA unwinding was conducted under pre-steady-state conditions, which favor binding of monomeric Dda. Under pre-steady-state conditions, the quantity of product decreased somewhat as the single-stranded length increased, from 12 to 24 nucleotides. Thus, when monomeric Dda is required to translocate longer distances prior to unwinding, processivity is lowered. Taken together, these results indicate that enhanced binding to the longer single-stranded overhangs was not responsible for enhanced processivity under conditions of excess enzyme. Rather, multiple molecules of Dda bound to the same substrate exhibit greater processivity for DNA unwinding.
先前已表明，Dda作为单体在解开DNA [Nanduri等人（2002年），《美国国家科学院院刊》99, 14722] 和置换链霉亲和素 [Byrd和Raney（2004年），《自然结构与分子生物学》11, 531] 方面具有活性。然而，其置换链霉亲和素的活性随着单链DNA长度的增加而增强。我们研究了Dda是否会随着单链突出端长度的增加，对部分双链DNA底物表现出增强的解旋作用。制备了含有16个碱基对以及4、6、8、12、16、20和24个核苷酸单链突出端的DNA底物。在存在过量酶的单轮反应条件下，随着单链突出端长度的增加，解开的DNA量显著增加。当DNA底物含有更长的单链突出端时，观察到持续性增加。平衡结合研究表明，Dda与含有较长突出端的底物的结合明显优于较短突出端的底物。为了确定解旋持续性的增加是由于多个Dda分子还是由于对较长突出端的结合亲和力增加，在有利于单体Dda结合的预稳态条件下进行了DNA解旋实验。在预稳态条件下，随着单链长度从12增加到24个核苷酸，产物量有所下降。因此，当单体Dda在解旋之前需要移动更长距离时，持续性会降低。综上所述，这些结果表明，在过量酶条件下，与较长单链突出端的增强结合并非导致增强持续性的原因。相反，结合到同一底物上的多个Dda分子在DNA解旋方面表现出更大的持续性
