Quantitative determination of uracil residues in Escherichia coli DNA:: Contribution of ung, dug, and dut genes to uracil avoidance
Quantitative determination of uracil residues in Escherichia coli DNA:: Contribution of ung, dug, and dut genes to uracil avoidance
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DOI：
10.1016/j.dnarep.2006.06.009
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发表时间：
2006-12-09
期刊：
影响因子：
3.8
通讯作者：
Bennett, Samuel E.
中科院分区：
文献类型：
--
作者：
Lari, Sibghat-Ullah;Chen, Cheny-Yao;Bennett, Samuel E.
测定了从大肠杆菌菌株中提取的DNA中尿嘧啶残基的稳态水平，并测定了尿嘧啶-DNA糖基化酶（ung）、双链尿嘧啶-DNA糖基化酶（dug）和dUTP焦磷酸酶（dut）基因缺陷对尿嘧啶蓄积的影响。开发了一种灵敏的方法，称为Ung-ARP测定，该方法利用E. coli Ung、T4 pdg和醛反应性探针试剂，以标记由生物素切除尿嘧啶产生的脱碱基位点。检测限为1个尿嘧啶残基/100万个核苷酸（U/10（6）nt）。尿嘧啶在大肠杆菌基因组DNA中的水平。大肠杆菌JM 105（ung（+）dug（+））中的突变体在检测限内，与同基因dug突变体一样，无论生长期如何。在JM 105中，ung的失活导致在对数生长早期为31 +/-2.6U/10（6）nt，在饱和期为19 +/-1.7U/10（6）nt。一个ungdug双突变体（CY 11）在对数生长早期和饱和生长期分别积累了33 +/- 2.9 U/10（6）nt和23 +/- 1.8 U/10（6）nt。当CY 11的培养物补充有20 ng/ml的5-氟-2 '-脱氧尿苷时，早期对数生长期DNA中的尿嘧啶水平上升至125 ± 1.7 U/106 nt。脱氧尿苷补充减少CY 11 DNA中尿嘧啶的量，但尿苷没有。通过Ung-ARP试验、代谢标记的P-32 DNA的二维薄层色谱法以及尿嘧啶和胸腺嘧啶脱氧核苷的LC/MS测定，从CJ 236（dut-1 ung-1）提取的DNA中尿嘧啶的水平确定为3000-8000 U/10（6）nt。DNA测序结果表明，唯一的分子缺陷在CJ 236，dUTP焦磷酸酶基因是一个C T转换突变，导致在一个Thr 24 Ile氨基酸的变化。(c)2006 Elsevier B. V.保留所有权利。
The steady-state levels of uracil residues in DNA extracted from strains of Escherichia coli were measured and the influence of defects in the genes for uracil-DNA glycosylase (ung), double-strand uracil-DNA glycosylase (dug), and dUTP pyrophosphatase (dut) on uracil accumulation was determined. A sensitive method, called the Ung-ARP assay, was developed that utilized E. coli Ung, T4pdg, and the Aldehyde Reactive Probe reagent to label abasic sites resulting from uracil excision with biotin. The limit of detection was one uracil residue per million DNA nucleotides (U/10(6) nt). Uracil levels in the genomic DNA of E. coli JM105 (ung(+) dug(+)) were at the limit of detection, as were those of an isogenic dug mutant, regardless of growth phase. Inactivation of ung in JM105 resulted in 31 +/- 2.6 U/10(6) nt during early log growth and 19 +/- 1.7 U/10(6) nt in saturated phase. An ung dug double mutant (CY11) accumulated 33 +/- 2.9 U/10(6) nt and 23 +/- 1.8 U/10(6) nt during early log and saturated phase growth, respectively. When cultures of CY11 were supplemented with 20 ng/ml of 5-fluoro-2'-deoxyuridine, uracil levels in early log phase growth DNA rose to 125 1.7 U/106 nt. Deoxyuridine supplementation reduced the amount of uracil in CY11 DNA, but uridine did not. Levels of uracil in DNA extracted from CJ236 (dut-1 ung-1) were determined to be 3000-8000 U/10(6) nt as measured by the Ung-ARP assay, two-dimensional thin-layer chromatography of metabolically-labeled P-32 DNA, and LC/MS of uracil and thymine deoxynucleosides. DNA sequencing revealed that the sole molecular defect in the CJ236, dUTP pyrophosphatase gene was a C T transition mutation that resulted in a Thr24Ile amino acid change. (c) 2006 Elsevier B.V. All rights reserved.