Kuznetsova Svetlana, Rykhlevskaya Anna, Taranenko Maria, Sidorkina Olga, Oretskaya Tatiana, Laval Jacques
Laboratory of Nucleic Acids Chemistry, Department of Chemistry, Moscow State University, Moscow 119899, Russia.
Biochimie. 2003 May;85(5):511-9. doi: 10.1016/s0300-9084(03)00067-1.
Specific contacts between DNA phosphate groups and positively charged nucleophilic amino acids from the Escherichia coli Fpg protein play a significant role in DNA-Fpg protein interaction. In order to identify these phosphate groups the chemical crosslinking procedure was carried out. The probing of the Fpg protein active center was performed using a series of reactive DNA duplexes containing both a single 7,8-dihydro-8-oxoguanosine (oxoG) residue and O-alkyl-substituted pyrophosphate internucleotide groups at the same time. Reactive internucleotide groups were introduced in dsDNA immediately 5' or 3' to the oxidative lesion and one or two nucleotides 5' or 3' away from it. We showed that the Fpg protein specifically binds to the modified DNA duplexes. The binding efficiency varied with the position of the reactive group and was higher for the duplexes containing substituted pyrophosphate groups at the ends of pentanucleotide with the oxoG in the center. The nicking efficiency of the DNA duplexes containing the reactive groups one or two nucleotides 5' away from the lesion was higher as compared to non-modified DNA duplex bearing only the oxidative damage. We found two novel non-hydrolizable substrate analogs for the Fpg protein containing pyrophosphate and substituted pyrophosphate groups 3' adjacent to the oxoG. Using crosslinking, we revealed the phosphate groups, 3' and 5' adjacent to the lesion, which have specific contacts with nucleophilic amino acids from the E. coli Fpg protein active center. The crosslinking efficiency achieved 30%. The approaches developed can be employed in the studies of pro- and eucaryotic homologs of the E. coli Fpg protein as well as other repair enzymes.
大肠杆菌Fpg蛋白中带正电荷的亲核氨基酸与DNA磷酸基团之间的特异性接触在DNA-Fpg蛋白相互作用中起着重要作用。为了鉴定这些磷酸基团，我们进行了化学交联实验。我们使用了一系列同时含有单个7,8-二氢-8-氧代鸟苷（oxoG）残基和O-烷基取代的焦磷酸核苷酸间基团的反应性DNA双链体，对Fpg蛋白的活性中心进行了探测。反应性核苷酸间基团被引入双链DNA中，紧挨着氧化损伤位点的5'端或3'端，以及距离该位点5'或3'方向的一两个核苷酸处。我们发现Fpg蛋白能特异性地结合到修饰后的DNA双链体上。结合效率随反应基团的位置而变化，对于中心含有oxoG的五核苷酸末端带有取代焦磷酸基团的双链体，其结合效率更高。与仅带有氧化损伤的未修饰DNA双链体相比，在损伤位点5'方向一两个核苷酸处含有反应基团的DNA双链体的切口效率更高。我们发现了两种新型的Fpg蛋白非水解性底物类似物，它们在oxoG的3'端含有焦磷酸和取代焦磷酸基团。通过交联实验，我们揭示了与损伤位点相邻的3'和5'磷酸基团，它们与大肠杆菌Fpg蛋白活性中心的亲核氨基酸有特异性接触。交联效率达到了30%。所开发的方法可用于研究大肠杆菌Fpg蛋白的原核和真核同源物以及其他修复酶。
