Gutiérrez J, Garmendia C, Salas M
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Nucleic Acids Res. 1988 Jul 11;16(13):5895-914. doi: 10.1093/nar/16.13.5895.
The origins of replication of phi 29 DNA have been studied by analyzing the activity as templates in the phi 29 in vitro replication system of E. coli recombinant plasmids and M13 derivatives containing phi 29 DNA terminal sequences. Plasmid pITR, containing the 6 bp long inverted terminal repeat of phi 29 DNA, was shown to be essentially inactive. The analysis of a series of deletion derivatives of plasmid pID13, that contains the 73 and 269 bp from the left and right phi 29 DNA ends, respectively, indicated that the minimal origins of replication are comprised within the mutagenesis at these sequences was carried out. Changes of the second or third A into a C completely abolished the template activity. In the case of changes at position from 4 to 12, only 3 out of 14 mutations reduced the template activity; these 3 mutations were double changes and 2 of them affected the inverted terminal repeat. The results suggest that the sequence requirement at the end-proximal region of the origin of replication is more strict than that at the distal region.
通过分析含有phi 29 DNA末端序列的大肠杆菌重组质粒和M13衍生物在phi 29体外复制系统中作为模板的活性，对phi 29 DNA的复制起点进行了研究。含有phi 29 DNA 6 bp长反向末端重复序列的质粒pITR基本上没有活性。对质粒pID13的一系列缺失衍生物进行分析，该质粒分别包含来自phi 29 DNA左端和右端的73 bp和269 bp，结果表明最小复制起点包含在这些序列中，并且对这些序列进行了诱变。将第二个或第三个A突变为C完全消除了模板活性。在位置4至12处发生变化的情况下，14个突变中只有3个降低了模板活性；这3个突变是双重变化，其中2个影响反向末端重复序列。结果表明，复制起点近端区域的序列要求比远端区域更严格。
