Hales L M, Gumport R I, Gardner J F
Department of Microbiology, University of Illinois, Urbana 61801, USA.
Biochimie. 1994;76(10-11):1030-40. doi: 10.1016/0300-9084(94)90027-2.
Integration host factor (IHF) is a protein encoded by Escherichia coli, which was first discovered as a requirement for bacteriophage lambda site-specific recombination. In this study, we characterized mutants of IHF for their ability to bind to various IHF binding sites in vivo and to promote recombination of lambda in vitro. DNA-binding in vivo was monitored using the challenge-phage system. If IHF binds to its DNA-binding site that has been placed into the P(ant) region of bacteriophage P22, it acts as a repressor of the ant (antirepressor) gene, leading to the formation of lysogens of Salmonella typhimurium. If IHF cannot bind to its site, antirepressor is made leading to cell lysis. Challenge phages containing chimeras of different lambda IHF binding sites were constructed to test the contribution to the binding of a dA+dT-rich region, found in the sequence of the H' site but not in the H' site. In one case, the binding of mutant IHF proteins was enhanced by the presence of the dA+dT-rich region, indicating that IHF may be affected by neighboring bases and local DNA structure when it binds to its site. A subset of the mutant proteins retained the ability to form a looped attL complex in vivo, representing part of a higher-order protein-DNA complex (the 'intasome'). Additionally, this same subset of proteins also promoted the integration and excision of bacteriophage lambda in vitro. Thus, these mutant proteins not only retain their DNA-bending ability but make any protein-protein contacts necessary to form a recombination-proficient intasome.
整合宿主因子（IHF）是一种由大肠杆菌编码的蛋白质，它最初是作为噬菌体λ位点特异性重组的必要条件被发现的。在本研究中，我们对IHF突变体进行了表征，以研究它们在体内与各种IHF结合位点结合的能力以及在体外促进λ重组的能力。使用挑战噬菌体系统监测体内的DNA结合情况。如果IHF与其已被置于噬菌体P22的P（ant）区域的DNA结合位点结合，它会作为抗阻遏物（antirepressor）基因的阻遏物起作用，导致鼠伤寒沙门氏菌溶原菌的形成。如果IHF不能与其位点结合，抗阻遏物就会产生，导致细胞裂解。构建了包含不同λ IHF结合位点嵌合体的挑战噬菌体，以测试富含dA + dT区域对结合的贡献，该区域存在于H'位点序列中但不存在于H位点序列中。在一种情况下，富含dA + dT区域的存在增强了突变体IHF蛋白的结合，这表明IHF在与其位点结合时可能会受到相邻碱基和局部DNA结构的影响。一部分突变蛋白在体内保留了形成环状attL复合物的能力，这代表了高阶蛋白质 - DNA复合物（“整合体”）的一部分。此外，同一部分蛋白质在体外也促进了噬菌体λ的整合和切除。因此，这些突变蛋白不仅保留了它们的DNA弯曲能力，还形成了形成具有重组能力的整合体所需的任何蛋白质 - 蛋白质接触。
