Functional interactions of mitochondrial DNA polymerase and single-stranded DNA-binding protein - Template-primer DNA binding and initiation and elongation of DNA strand synthesis
Functional interactions of mitochondrial DNA polymerase and single-stranded DNA-binding protein - Template-primer DNA binding and initiation and elongation of DNA strand synthesis
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DOI：
10.1074/jbc.274.21.14779
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发表时间：
1999-05-21
影响因子：
4.8
通讯作者：
Kaguni, LS
中科院分区：
文献类型：
--
作者：
Farr, CL;Wang, YX;Kaguni, LS
线粒体DNA聚合酶（poly γ）和线粒体单链DNA结合蛋白（mtSSB）从果蝇胚胎之间的功能相互作用进行了评估方面的总体活动poly γ和部分反应，涉及模板引物结合和启动和闲置的DNA链合成。pol γ中的5' -> 3' DNA聚合酶和3' -> 5'核酸外切酶都被天然和重组形式的mtSSB刺激15-20倍。在广泛的KCl浓度范围内，两种酶活性的刺激程度相似，表明它们的功能协调和类似的mtSSB刺激机制。与此同时，在核酸外切酶解水解中保持了聚合酶γ的高错配特异性，表明聚合酶γ催化效率的增强可能不伴随核苷酸周转的增加。聚合物γ DNA复合物的DNA酶I足迹和初始速率测量表明，mtSSB增强引物识别和结合，并刺激30倍的DNA链起始速率。解离研究表明，天然聚γ异二聚体与模板引物DNA的生产复合物形成，并保持稳定的复制辅助蛋白的情况下。
Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single stranded DNA-binding protein (mtSSB) from Drosophila embryos have been evaluated with regard to the overall activity of poli gamma and in partial reactions involving template-primer binding and initiation and idling in DNA strand synthesis. Both the 5' --> 3' DNA polymerase and 3' --> 5' exonuclease in pol gamma are stimulated 15-20-fold on oligonucleotide-primed single-stranded DNA by native and recombinant forms of mtSSB. That the extent of stimulation is similar for both enzyme activities over a broad range of KCI concentrations suggests their functional coordination and a similar mechanism of stimulation by mtSSB. At the same time, the high mispair specificity of pol gamma in exonucleolytic hydrolysis is maintained, indicating that enhancement of pol gamma catalytic efficiency is likely not accompanied by increased nucleotide turnover. DNase I footprinting of pol gamma DNA complexes and initial rate measurements show that mtSSB enhances primer recognition and binding and stimulates 30-fold the rate of initiation of DNA strands. Dissociation studies show that productive complexes of the native pol gamma heterodimer with template-primer DNA are formed and remain stable in the absence of replication accessory proteins.