Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Tuberculosis (Edinb). 2011 May;91(3):212-8. doi: 10.1016/j.tube.2011.02.001. Epub 2011 Mar 2.
Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria.
尿嘧啶切除修复在所有生命领域中普遍存在，由尿嘧啶 DNA 糖基化酶（UDG）启动，UDG 从 DNA 中切除诱变碱基尿嘧啶，留下无碱基位点（AP 位点）。产生的 AP 位点的修复需要 AP 内切酶、DNA 聚合酶和 DNA 连接酶，它们的联合活性导致短补丁或长补丁修复。结核分枝杆菌是结核病的病原体，由于其富含 G+C 的基因组以及在宿主巨噬细胞内的生态位，使其面临活性氮和氧物种的暴露，这是 DNA 中胞嘧啶脱氨（变成尿嘧啶）的两个主要原因，因此其积累尿嘧啶的风险增加。在体外研究这种重要人类病原体的 DNA 修复的实验受到限制。为了研究分枝杆菌中的尿嘧啶切除修复，我们使用结核分枝杆菌和耻垢分枝杆菌（一种快速生长的分枝杆菌）的无细胞提取物以及寡聚体或质粒 DNA 底物建立了测定条件。我们表明，在分枝杆菌中，尿嘧啶切除修复主要通过长补丁修复完成。此外，我们还表明，新鉴定的家族 5 UDG，分枝杆菌 UDG B，可替代高度保守的家族 1 UDG，UNG，这表明 UDG B 可能在分枝杆菌的尿嘧啶切除修复中充当备用酶。
