Handa P, Acharya N, Varshney U
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India.
J Biol Chem. 2001 May 18;276(20):16992-7. doi: 10.1074/jbc.M100393200. Epub 2001 Feb 27.
Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E. coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG. In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E. coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs. The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG. The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG. The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.
尿嘧啶是DNA中的一种促诱变碱基，可通过胞嘧啶的自发脱氨作用或DNA聚合酶掺入dUMP而产生。尿嘧啶DNA糖基化酶（UDG）可将尿嘧啶从DNA中去除，它是尿嘧啶切除修复途径中的首个酶。我们最近报道，大肠杆菌单链DNA结合蛋白（SSB）促进了大肠杆菌UDG（EcoUDG）从某些结构化底物中切除尿嘧啶，并提示SSB与UDG之间存在相互作用。在本研究中，我们利用通过融合大肠杆菌和结核分枝杆菌的SSB的N端和C端结构域获得的嵌合蛋白，来研究SSB与UDG之间的相互作用。EcoSSB或包含其C端结构域的嵌合体在二元（SSB-UDG）或三元（DNA-SSB-UDG）复合物中与EcoUDG相互作用。然而，包含EcoSSB N端结构域的嵌合体与EcoUDG没有相互作用。因此，EcoSSB的C端结构域（48个氨基酸）对于与EcoUDG相互作用是必要且充分的。数据还表明，MtuSSB的C端结构域（34个氨基酸）是介导其与MtuUDG相互作用的主要决定因素。文中讨论了SSB与UDG之间的相互作用在尿嘧啶切除修复途径中如何发挥重要作用的机制。
