Guzzo A, Lee M H, Oda K, Walker G C
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
J Bacteriol. 1996 Dec;178(24):7295-303. doi: 10.1128/jb.178.24.7295-7303.1996.
On the basis of characterizations of a set of UmuD monocysteine derivatives, we had suggested that positions 24, 34, and 44 are closer to the intact UmuD homodimer interface than other positions tested (M. H. Lee, T. Ohta, and G. C. Walker, J. Bacteriol. 176:4825-4837, 1994). Because this region of UmuD also appeared to be important for interactions with RecA, we followed up on our previous study by constructing a second set of monocysteine UmuD derivatives with single cysteine substitutions at positions 30 to 42. We found that like the VC34 mutant, UmuD derivatives with monocysteine substitutions at positions 32 and 35 showed deficiencies in in vivo and in vitro RecA-mediated cleavage as well as in UV mutagenesis, suggesting that the position 32 to 35 region may be important for RecA-mediated cleavage of UmuD. Interestingly, UmuD with monocysteine substitutions at residues 33 and 40 showed a reduction in UV mutagenesis while retaining the ability to be cleaved by RecA in vivo, suggesting a deficiency in the subsequent role of the UmuD' derivatives in mutagenesis. All of the UmuD monocysteine derivatives in the position 30 to 42 series purified indistinguishably from the wild-type protein. The observations that purified proteins of the UmuD derivatives RC37 and IC38 could be disulfide cross-linked quantitatively upon addition of iodine and yet were poorly modified with iodoacetate led us to suggest that the pairs of residues at positions 37 and 38 are extremely close to the UmuD2 homodimer interface. These observations indicate that the structure of the UmuD2 homodimer in solution is very different from the crystal structure of the UmuD'2 homodimer reported by Peat et al. (T. S. Peat, E. G. Frank, J. P. McDonald, A. S. Levine, R. Woodgate, and W. A. Hendrickson, Nature [London] 380:727-730, 1996).
基于一组UmuD单半胱氨酸衍生物的特性，我们曾提出，与其他测试位置相比，24、34和44位更靠近完整的UmuD同型二聚体界面（M. H. Lee、T. Ohta和G. C. Walker，《细菌学杂志》176:4825 - 4837，1994年）。由于UmuD的这一区域对于与RecA的相互作用似乎也很重要，我们通过构建第二组在30至42位有单个半胱氨酸取代的单半胱氨酸UmuD衍生物来跟进我们之前的研究。我们发现，与VC34突变体一样，在32和35位有单半胱氨酸取代的UmuD衍生物在体内和体外RecA介导的切割以及紫外线诱变方面都存在缺陷，这表明32至35位区域可能对RecA介导的UmuD切割很重要。有趣的是，在33和40位有单半胱氨酸取代的UmuD在紫外线诱变方面有所降低，同时在体内保留了被RecA切割的能力，这表明UmuD'衍生物在诱变中的后续作用存在缺陷。30至42位系列中的所有UmuD单半胱氨酸衍生物与野生型蛋白在纯化时没有区别。纯化的UmuD衍生物RC37和IC38蛋白在加入碘后可以定量地进行二硫键交联，但用碘乙酸修饰效果不佳，这一观察结果使我们提出，37和38位的残基对非常靠近UmuD2同型二聚体界面。这些观察结果表明，溶液中UmuD2同型二聚体的结构与Peat等人报道的UmuD'2同型二聚体的晶体结构非常不同（T. S. Peat、E. G. Frank、J. P. McDonald、A. S. Levine、R. Woodgate和W. A. Hendrickson，《自然》[伦敦]380:727 - 730，1996年）。
