Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, 630090 Novosibirsk, Russia.
Biochemistry (Mosc). 2012 Apr;77(4):346-53. doi: 10.1134/S0006297912040050.
We have examined the influence of centrin 2 (Cen2) on the interaction of nucleotide excision repair factors (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes bearing the dUMP derivative 5-{3-[6-(carboxyamidofluoresceinyl)amidocapromoyl]allyl}-2'-deoxyuridine-5'-monophosphate. The fluorescein residue linked to the nucleotide base imitates a bulky lesion of DNA. Cen2 stimulated the binding and increased the yield of DNA adducts with XPC-HR23b, a protein recognizing bulky damages in DNA. Stimulation of the binding was most pronounced in the presence of Mg(2+) and demonstrated a bell-shaped dependence on Cen2 concentration. The addition of Cen2 changed the stoichiometry of RPA-DNA complexes and diminished the yield of RPA-DNA covalent crosslinks. We have shown that Cen2 influences the binding of RPA and XPA with DNA, which results in formation of additional DNA-protein complexes possibly including Cen2. We have also found some evidence of direct contacts between Cen2 and DNA. These results in concert with the literature data suggest that Cen2 can be a regulatory element in the nucleotide excision repair system.
我们研究了中心体蛋白 2（Cen2）对核苷酸切除修复因子（XPC-HR23b、RPA 和 XPA）与含有 dUMP 衍生物 5-{3-[6-(羧基荧光素酰胺基)卡普罗米酰基]烯丙基}-2'-脱氧尿苷-5'-单磷酸的 48 -mer DNA 双链体相互作用的影响。连接到核苷酸碱基上的荧光素残基模拟了 DNA 的大体积损伤。Cen2 刺激了与 XPC-HR23b 的结合，并增加了 DNA 加合物的产量，XPC-HR23b 是一种识别 DNA 中大体积损伤的蛋白质。在存在 Mg2+的情况下，结合的刺激最为明显，并表现出对 Cen2 浓度的钟形依赖性。添加 Cen2 改变了 RPA-DNA 复合物的化学计量，并降低了 RPA-DNA 共价交联的产量。我们已经表明，Cen2 影响 RPA 和 XPA 与 DNA 的结合，这导致形成可能包括 Cen2 的额外 DNA-蛋白质复合物。我们还发现了 Cen2 与 DNA 之间存在直接接触的一些证据。这些结果与文献数据一起表明，Cen2 可以是核苷酸切除修复系统的调节元件。
