Purnapatre K, Handa P, Venkatesh J, Varshney U
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Nucleic Acids Res. 1999 Sep 1;27(17):3487-92. doi: 10.1093/nar/27.17.3487.
Deamination of cytosines results in accumulation of uracil residues in DNA, which unless repaired lead to GC-->AT transition mutations. Uracil DNA glyco-sylase excises uracil residues from DNA and initiates the base excision repair pathway to safeguard the genomic integrity. In this study, we have investigated the effect of single-stranded DNA binding proteins (SSBs) from Escherichia coli (Eco SSB) and Mycobacterium tuberculosis (Mtu SSB) on uracil excision from synthetic substrates by uracil DNA glycosylases (UDGs) from E. coli, Mycobacterium smegmatis and M.tuberculosis (referred to as Eco -, Msm - and Mtu UDGs respectively). Presence of SSBs with all the three UDGs resulted in decreased efficiency of uracil excision from a single-stranded 'unstructured' oligonucleo-tide, SS-U9. On the other hand, addition of Eco SSB to Eco UDG, or Mtu SSB to Mtu UDG reactions resulted in increased efficiency of uracil excision from a hairpin oligonucleotide containing dU at the second position in a tetraloop (Loop-U2). Interestingly, the efficiency of uracil excision by Msm UDG from the same substrate was decreased in the presence of either Eco- or Mtu SSBs. Furthermore, Mtu SSB also decreased uracil excision from Loop-U2 by Eco UDG. Our studies using surface plasmon resonance technique demonstrated interactions between the homologous combinations of SSBs and UDGs. Heterologous combinations either did not show detectable interaction (Eco SSB with Mtu UDG) or showed a relatively weaker interaction (Mtu SSB with Eco UDG). Taken together, our studies suggest differential interactions between the two groups (SSBs and UDGs) of the highly conserved proteins. Such studies may provide important clues to design selective inhibitors against this important class of DNA repair enzymes.
胞嘧啶脱氨基会导致DNA中尿嘧啶残基的积累，若不修复，会导致GC→AT转换突变。尿嘧啶DNA糖基化酶从DNA中切除尿嘧啶残基，并启动碱基切除修复途径以维护基因组完整性。在本研究中，我们研究了来自大肠杆菌（Eco SSB）和结核分枝杆菌（Mtu SSB）的单链DNA结合蛋白对大肠杆菌、耻垢分枝杆菌和结核分枝杆菌（分别称为Eco -、Msm -和Mtu UDGs）的尿嘧啶DNA糖基化酶从合成底物中切除尿嘧啶的影响。所有三种UDGs与SSBs同时存在会导致从单链“无结构”寡核苷酸SS-U9中切除尿嘧啶的效率降低。另一方面，在Eco UDG反应中添加Eco SSB，或在Mtu UDG反应中添加Mtu SSB，会导致从四环中第二个位置含有dU的发夹寡核苷酸（Loop-U2）中切除尿嘧啶的效率提高。有趣的是，在存在Eco -或Mtu SSBs的情况下，Msm UDG从同一底物中切除尿嘧啶 的效率降低。此外，Mtu SSB也降低了Eco UDG从Loop-U2中切除尿嘧啶的效率。我们使用表面等离子体共振技术的研究证明了SSBs和UDGs同源组合之间的相互作用。异源组合要么未显示可检测到的相互作用（Eco SSB与Mtu UDG），要么显示相对较弱的相互作用（Mtu SSB与Eco UDG）。综上所述，我们的研究表明这两类高度保守的蛋白质（SSBs和UDGs）之间存在差异相互作用。此类研究可能为设计针对这类重要DNA修复酶的选择性抑制剂提供重要线索。
