McLenigan M P, Kulaeva O I, Ennis D G, Levine A S, Woodgate R
Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2725, USA.
J Bacteriol. 1999 Nov;181(22):7005-13. doi: 10.1128/JB.181.22.7005-7013.1999.
The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD'. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD' protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD'-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD' functions in vivo as well as examined HumD's physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive DeltaumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD', HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD'-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo.
大肠杆菌的umuD和umuC基因组成一个操纵子，编码参与对正常情况下抑制复制的DNA损伤进行诱变旁路的蛋白质。然而，UmuD在经历RecA介导的切割反应生成UmuD'之前，无法在这个过程中发挥作用。现在已经鉴定出许多umuDC的同源物。大多数位于细菌染色体或广宿主范围的R质粒上。然而，一种这样的假定同源物humD（umuD的同源物）却存在于噬菌体P1基因组上。有趣的是，humD与其他umuD同源物的不同之处在于，它编码的蛋白质大小与翻译后生成的UmuD'蛋白相似，而不是UmuD，并且它也不在与同源umuC伴侣组成的操纵子中。为了确定HumD实际上是否是原核UmuD'样诱变蛋白的真正同源物，我们分析了HumD在体内补充UmuD'功能的能力，并在体外检测了HumD的物理特性。当从高拷贝数质粒表达时，HumD分别恢复了细胞诱变作用，并提高了正常情况下不可诱变的recA430 lexA(Def)和对紫外线敏感的DeltaumuDC recA718 lexA(Def)菌株的紫外线存活率。当从低拷贝数质粒表达HumD时，互补活性降低，但免疫分析解释了这一观察结果，即HumD在体内通常表达不佳。体外分析表明，与UmuD'一样，HumD在溶液中形成稳定的二聚体，并且能够与大肠杆菌UmuC和RecA核蛋白丝相互作用。因此，我们得出结论，噬菌体P1 HumD是UmuD'样蛋白的功能同源物，并且我们推测了P1在体内可能需要这种蛋白活性的原因。
