Gong Chunling, Martins Alexandra, Bongiorno Paola, Glickman Michael, Shuman Stewart
Molecular Biology and Immunology Programs, Sloan-Kettering Institute, and Infectious Disease Division, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
J Biol Chem. 2004 May 14;279(20):20594-606. doi: 10.1074/jbc.M401841200. Epub 2004 Feb 25.
Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.
结核分枝杆菌编码一种依赖NAD⁺的DNA连接酶（LigA）以及三种不同的依赖ATP的连接酶同源物（LigB、LigC和LigD）。在此，我们纯化并表征了分枝杆菌的多种DNA连接酶，并通过遗传学方法探究依赖ATP的连接酶对于结核分枝杆菌生长是否必需。我们发现分枝杆菌连接酶与带切口的DNA底物的反应性存在显著差异，其中LigA和LigB分别在NAD⁺和ATP存在时表现出强烈的切口封闭活性，而具有ATP特异性腺苷酸转移酶活性的LigC和LigD则表现出较弱的切口连接活性并产生高水平的DNA - 腺苷酸中间体。所有四种分枝杆菌连接酶均为单体酶。LigA对NAD⁺的Kₘ值较低（1微摩尔），并且对最近描述的一种依赖NAD⁺的连接酶的吡啶并色满酮抑制剂敏感。LigA能够替代必需的酵母连接酶Cdc9维持酿酒酵母的生长，但LigB、LigC和LigD则不能。LigB的特点是其对ATP的Kₘ值相对较高（0.34毫摩尔），并且其切口连接依赖于一个独特的N端结构域。三种依赖ATP的连接酶对分枝杆菌生长均非必需。结核分枝杆菌ligD缺失细胞在非同源DNA末端连接方面存在缺陷。
