Heltzel Justin M H, Maul Robert W, Scouten Ponticelli Sarah K, Sutton Mark D
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, 3435 Main Street, 140 Farber Hall, Buffalo, NY 14214, USA.
Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):12664-9. doi: 10.1073/pnas.0903460106. Epub 2009 Jul 16.
The actions of Escherichia coli DNA Polymerase IV (Pol IV) in mutagenesis are managed by its interaction with the beta sliding clamp. In the structure reported by Bunting et al. [EMBO J (2003) 22:5883-5892], the C-tail of Pol IV contacts a hydrophobic cleft on the clamp, while residues V303-P305 reach over the dimer interface to contact the rim of the adjacent clamp protomer. Using mutant forms of these proteins impaired for either the rim or the cleft contacts, we determined that the rim contact was dispensable for Pol IV replication in vitro, while the cleft contact was absolutely required. Using an in vitro assay to monitor Pol III*-Pol IV switching, we determined that a single cleft on the clamp was sufficient to support the switch, and that both the rim and cleft contacts were required. Results from genetic experiments support a role for the cleft and rim contacts in Pol IV function in vivo. Taken together, our findings challenge the toolbelt model and suggest instead that Pol IV contacts the rim of the clamp adjacent to the cleft that is bound by Pol III* before gaining control of the same cleft that is bound by Pol III*.
大肠杆菌DNA聚合酶IV（Pol IV）在诱变中的作用是通过其与β滑动夹的相互作用来调控的。在Bunting等人报道的结构中[《欧洲分子生物学组织杂志》（2003年）22:5883 - 5892]，Pol IV的C末端与夹上的一个疏水裂缝接触，而残基V303 - P305延伸至二聚体界面上方以接触相邻夹亚基的边缘。使用这些蛋白质的突变形式，其边缘或裂缝接触受损，我们确定边缘接触对于体外Pol IV复制是可有可无的，而裂缝接触是绝对必需的。使用体外测定法监测Pol III* - Pol IV转换，我们确定夹上的单个裂缝足以支持转换，并且边缘和裂缝接触都是必需的。遗传实验结果支持裂缝和边缘接触在体内Pol IV功能中的作用。综上所述，我们的发现挑战了工具带模型，相反表明在控制由Pol III结合的相同裂缝之前，Pol IV接触与由Pol III结合的裂缝相邻的夹边缘。
