Maul Robert W, Ponticelli Sarah K Scouten, Duzen Jill M, Sutton Mark D
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, SUNY, Buffalo, New York 14214, USA.
Mol Microbiol. 2007 Aug;65(3):811-27. doi: 10.1111/j.1365-2958.2007.05828.x.
Escherichia coli strains expressing the mutant beta159-sliding clamp protein (containing both a G66E and a G174A substitution) are temperature sensitive for growth and display altered DNA polymerase (pol) usage. We selected for suppressors of the dnaN159 allele able to grow at 42 degrees C, and identified four intragenic suppressor alleles. One of these alleles (dnaN780) contained only the G66E substitution, while a second (dnaN781) contained only the G174A substitution. Genetic characterization of isogenic E. coli strains expressing these alleles indicated that certain phenotypes were dependent upon only the G174A substitution, while others required both the G66E and G174A substitutions. In order to understand the individual contributions of the G66E and the G174A substitution to the dnaN159 phenotypes, we utilized biochemical approaches to characterize the purified mutant beta159 (G66E and G174A), beta780 (G66E) and beta781 (G174A) clamp proteins. The G66E substitution conferred a more pronounced effect on pol IV replication than it did pol II or pol III, while the G174A substitution conferred a greater effect on pol III and pol IV than it did pol II. Taken together, these findings indicate that pol II, pol III and pol IV interact with distinct, albeit overlapping surfaces of the beta clamp.
表达突变型β159滑动夹蛋白（含有G66E和G174A双位点取代）的大肠杆菌菌株对温度敏感，生长受限，且显示出DNA聚合酶（pol）使用情况的改变。我们筛选出了能够在42℃生长的dnaN159等位基因的抑制子，并鉴定出四个基因内抑制子等位基因。其中一个等位基因（dnaN780）仅含有G66E取代，而另一个（dnaN781）仅含有G174A取代。对表达这些等位基因的同基因大肠杆菌菌株进行遗传特征分析表明，某些表型仅取决于G174A取代，而其他表型则需要G66E和G174A双位点取代。为了了解G66E和G174A取代对dnaN159表型的各自贡献，我们采用生化方法对纯化的突变型β159（G66E和G174A）、β780（G66E）和β781（G174A）夹蛋白进行了表征。G66E取代对pol IV复制的影响比对pol II或pol III的影响更为显著，而G174A取代对pol III和pol IV的影响比对pol II的影响更大。综上所述，这些发现表明pol II、pol III和pol IV与β夹的不同表面相互作用，尽管存在重叠。
