Zheng W, Li Z, Skarstad K, Crooke E
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington DC 20007, USA.
EMBO J. 2001 Mar 1;20(5):1164-72. doi: 10.1093/emboj/20.5.1164.
Cell growth arrests when the concentrations of anionic phospholipids drop below a critical level in Escherichia coli, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA-dependent initiation of DNA replication at the chromosomal origin (oriC). Mutations have been introduced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA-acidic phospholipid interactions. Expression of DnaA proteins possessing certain small deletions or substituted amino acids restored growth to cells deficient in acidic phospholipids, whereas expression of wild-type DnaA did not. The mutations include substitutions and deletions in the phospholipid-interacting domain as well as some small deletions in the DNA-binding domain of DnaA. Marker frequency analysis indicated that initiation of replication occurs at or near oriC in acidic phospholipid- deficient cells rescued by the expression of DnaA having a point mutation in the membrane-binding domain, DnaA(L366K). Flow cytometry revealed that expression in wild-type cells of plasmid-borne DnaA(L366K) and DnaA(Delta363-367) reduced the frequency with which replication was initiated and disturbed the synchrony of initiations.
在大肠杆菌中,当阴离子磷脂浓度降至临界水平以下时,细胞生长会停滞,酸性磷脂含量不足会对依赖DnaA的染色体复制起点(oriC)处的DNA复制起始产生不利影响。已将突变引入DnaA的羧基区域,包括被确定为对体外DnaA-酸性磷脂有效相互作用至关重要的部分。表达具有某些小缺失或取代氨基酸的DnaA蛋白可使缺乏酸性磷脂的细胞恢复生长,而野生型DnaA的表达则不能。这些突变包括磷脂相互作用结构域中的取代和缺失以及DnaA的DNA结合结构域中的一些小缺失。标记频率分析表明,在通过表达膜结合结构域中具有点突变的DnaA(DnaA(L366K))拯救的酸性磷脂缺陷细胞中,复制起始发生在oriC或其附近。流式细胞术显示,质粒携带的DnaA(L366K)和DnaA(Delta363-367)在野生型细胞中的表达降低了复制起始的频率,并扰乱了起始的同步性。