Vadi H V
Carcinogenesis. 1983 Nov;4(11):1379-84. doi: 10.1093/carcin/4.11.1379.
Plasmid pBR322 was alkylated with either chlorozotocin or with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene (BPDE) before it was transformed into various strains of Escherichia coli. Plasmid survival was determined as ability to convert the bacteria to tetracycline and ampicillin resistance. Increased levels of alkylation caused a decrease in transforming activity in all strains studied. This decrease did not seem to be a result of alkylation induced strand scission, but rather some other biochemical or conformational change induced by the alkylating event. In E. coli AB1157 transformation was decreased by 50% with 6 alkylations/plasmid molecule for BPDE and 8-9 alkylations for chlorozotocin. At these levels of alkylation the loss in supercoiled DNA due to strand scission was less than 5%. Alkylated pBR322 was also transformed into repair-deficient strains of E. coli. In strain JC2924 (recA6) the survival of both BPDE- and chlorozotocin-modified DNA was similar to survival in the repair proficient strain AB1157, which would indicate that postreplicational repair of BPDE- or chlorozotocin-modified plasmid DNA was not significant under these conditions. Chlorozotocin-modified pBR322 did not seem to be repaired by the bacterial uvr-endonucleases as determined by plasmid survival in strains AB1884 (uvrC34), AB1885 (uvrB5) and AB1886 (uvrA6). With BPDE-alkylated plasmid DNA the results were strikingly different. Strains AB1884 and AB1886 were more sensitive to BPDE modified DNA than the wild type strain AB1157. Strain AB1885 was similar to AB1157 in sensitivity to BPDE-alkylated plasmid. These findings suggest that bacterial uvr-endonucleases may be able to recognize and repair BPDE-alkylated pBR322. The role of the uvrB protein in repair of alkylated DNA needs to be further investigated.
在将质粒pBR322转化到各种大肠杆菌菌株之前，先用氯乙亚硝脲或r-7,t-8-二羟基-t-9,10-环氧-7,8,9,10-四氢苯并[a]芘（BPDE）对其进行烷基化处理。通过质粒将细菌转化为对四环素和氨苄青霉素耐药的能力来测定质粒的存活率。在所研究的所有菌株中，烷基化水平的提高导致转化活性降低。这种降低似乎不是烷基化诱导的链断裂的结果，而是烷基化事件诱导的其他一些生化或构象变化的结果。在大肠杆菌AB1157中，对于BPDE，每个质粒分子有6次烷基化，对于氯乙亚硝脲有8 - 9次烷基化时，转化活性降低了50%。在这些烷基化水平下，由于链断裂导致的超螺旋DNA损失小于5%。烷基化的pBR322也被转化到大肠杆菌的修复缺陷菌株中。在JC2924（recA6）菌株中，BPDE和氯乙亚硝脲修饰的DNA的存活率与修复 proficient 菌株AB1157中的存活率相似，这表明在这些条件下，BPDE或氯乙亚硝脲修饰的质粒DNA的复制后修复并不显著。通过在AB1884（uvrC34）、AB1885（uvrB5）和AB1886（uvrA6）菌株中的质粒存活率测定，氯乙亚硝脲修饰的pBR322似乎不能被细菌的uvr内切核酸酶修复。对于BPDE烷基化的质粒DNA，结果显著不同。AB1884和AB1886菌株对BPDE修饰的DNA比野生型菌株AB1157更敏感。AB1885菌株对BPDE烷基化质粒的敏感性与AB1157相似。这些发现表明细菌的uvr内切核酸酶可能能够识别并修复BPDE烷基化的pBR322。uvrB蛋白在烷基化DNA修复中的作用需要进一步研究。
