Skorupski K, Sauer B, Sternberg N
DuPont Merck Pharmaceutical Co. Glenolden Laboratory, PA 19036.
J Mol Biol. 1994 Oct 21;243(2):268-82. doi: 10.1006/jmbi.1994.1653.
The PacA and PacB subunits of the bacteriophage P1 DNA packaging enzyme (pacase) are necessary for cleavage of the phage packaging site (pac). In the accompanying paper, we show that the PacA subunit of the enzyme specifically binds to pac in the absence of PacB, but requires factors present in an Escherichia coli extract to do so. We show here that either of two E. coli DNA binding proteins, integration host factor (IHF) or HU, can replace this extract and promote the binding of PacA to pac. IHF binds to pac independently of PacA and DNase I footprinting experiments show that IHF protects approximately 40 bp of DNA around an IHF consensus sequence adjacent to the cleavage site. DNase I footprinting experiments also show that in the presence of either IHF or HU, PacA binds to the hexanucleotide sequences (5'-TGATCA/G) that flank the cleavage site and that have been previously shown to be essential for pac cleavage. The importance of IHF and HU in pac cleavage is further demonstrated by the severe reduction in both the fidelity and efficiency of pac cleavage in vitro with extracts lacking both IHF and HU. Addition of either IHF or HU to the deficient extracts renders them fully proficient for pac cleavage. Finally, we show that IHF bends DNA at the IHF site within pac. Based on these results, we propose a model that can account for the role of the various phage and host proteins, and for DNA bending in the pac cleavage reaction.
噬菌体P1 DNA包装酶（pacase）的PacA和PacB亚基对于噬菌体包装位点（pac）的切割是必需的。在随附的论文中，我们表明该酶的PacA亚基在没有PacB的情况下特异性结合pac，但需要大肠杆菌提取物中存在的因子才能做到这一点。我们在此表明，两种大肠杆菌DNA结合蛋白中的任何一种，即整合宿主因子（IHF）或HU，都可以替代这种提取物并促进PacA与pac的结合。IHF独立于PacA与pac结合，DNase I足迹实验表明，IHF保护切割位点附近与IHF共有序列相邻的约40 bp DNA。DNase I足迹实验还表明，在存在IHF或HU的情况下，PacA与切割位点两侧的六核苷酸序列（5'-TGATCA/G）结合，这些序列先前已被证明对pac切割至关重要。缺乏IHF和HU的提取物在体外pac切割的保真度和效率都严重降低，这进一步证明了IHF和HU在pac切割中的重要性。向缺陷提取物中添加IHF或HU可使它们完全具备pac切割能力。最后，我们表明IHF在pac内的IHF位点处使DNA弯曲。基于这些结果，我们提出了一个模型，该模型可以解释各种噬菌体和宿主蛋白的作用以及pac切割反应中DNA弯曲的作用。
