Weiss R B, Mineura K, Henderson E E, Duker N J, deRiel J K
Biochemistry. 1983 Sep 13;22(19):4501-7. doi: 10.1021/bi00288a023.
Uracil can occur in DNA either as a result of utilization of dUTP by DNA polymerases or from in situ deamination of cytosine. The latter results in transition mutations following the next round of replication. We describe a technique for the detection of uracil in DNA by a modification of the Maxam-Gilbert sequencing procedure. Reaction of end-labeled DNA with uracil-DNA glycosylase followed by 1 M piperidine results in scission products corresponding to locations of uracils. These are detected by autoradiography following electrophoresis through a sequencing gel. Comparison of these scission products with the DNA sequences elucidates the mechanism of origin of the DNA uracils. The technique was tested with a cloned human DNA sequence grown in a dut-,ung-strain of Escherichia coli, which incorporates uracil in place of thymine in its DNA, and by chemical deamination of cytosines in that sequence. The technique was expanded by use of alkaline and enzymic probes to investigate possible accumulation of uracil, base losses, and other modifications in human liver and brain DNA. No damaged DNA moieties were detected. This method is applicable to the study of any recoverable reiterated sequence by any enzyme preparation that can recognize modifications in DNA.
尿嘧啶可因DNA聚合酶利用dUTP或胞嘧啶原位脱氨而存在于DNA中。后者会在下一轮复制后导致转换突变。我们描述了一种通过改进Maxam-Gilbert测序程序来检测DNA中尿嘧啶的技术。末端标记的DNA与尿嘧啶-DNA糖基化酶反应，随后用1 M哌啶处理，会产生与尿嘧啶位置相对应的断裂产物。通过测序凝胶电泳后进行放射自显影来检测这些产物。将这些断裂产物与DNA序列进行比较，可阐明DNA中尿嘧啶的起源机制。该技术用在dut-、ung-大肠杆菌菌株中生长的克隆人DNA序列进行了测试，该菌株在其DNA中用尿嘧啶取代胸腺嘧啶，并对该序列中的胞嘧啶进行化学脱氨。通过使用碱性和酶促探针扩展了该技术，以研究人肝脏和脑DNA中尿嘧啶的可能积累、碱基丢失及其他修饰。未检测到受损的DNA部分。该方法适用于通过任何能识别DNA修饰的酶制剂对任何可回收的重复序列进行研究。
