荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作用  

Study on The Interactions of Bloom Helicase Catalytic Core With Double-Stranded DNA by Fluorescence Polarization Technology

作　　者：骆衡[1,2,3] 许厚强[1,2] 陈祥[1,2] 刘朝前[1,2] 许庆贺[1,2] 李坤[1,2] 

机构地区：[1]贵州大学动物科学学院,高原山地动物遗传育种与繁殖省部共建教育部重点实验室,贵阳550025 [2]贵州大学动物科学学院,贵州省动物遗传育种与繁殖重点实验室,贵阳550025 [3]贵州大学农学院,贵州省山地农业病虫害重点实验室,贵阳550025

出　　处：《生物化学与生物物理进展》2013年第3期255-265,共11页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展计划(973)资助项目(2010CB534912);教育部博士点基金(200806570003);贵州省优秀人才省长资金(200822);贵州省国际合作项目(黔科合外G字[2011]7008号);贵州大学研究生创新基金(农科2012027)资助项目~~

摘　　要：Bloom综合症(BLM)解旋酶是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要的作用.BLM解旋酶的突变可导致Bloom综合症,患者遗传不稳定易患多种类型癌症.本研究运用荧光偏振技术研究BLM解旋酶催化核心(BLM642～1290)与双链DNA(dsDNA)的相互作用,分析其相关特征参数,了解BLM642～1290解旋酶与dsDNA的结合和解链特性.结果表明:BLM642～1290解旋酶与dsDNA的结合和解链与dsDNA 3′端的单链DNA(ssDNA)长度有关;解旋酶优先结合于dsDNA底物的ssDNA末端,且每分子解旋酶可结合9.6 nt的ssDNA;dsDNA 3′端ssDNA的长度为9.6 nt时,解旋酶的解链效率达到最大且不再随其长度而变化.另外,BLM642～1290解旋酶也能够结合和解链钝末端dsDNA,但其结合亲和力和解链效率低于有3′端ssDNA的dsDNA.推测BLM642～1290解旋酶在与dsDNA底物结合和解链时是单体形式,可能以尺蠖的形式解开dsDNA.这些结果可为进一步研究BLM解旋酶的功能特征提供理论基础.Bloom syndrome helicase （BLM） is an important member of RecQ family of DNA helicases. It participates in cell metabolism including DNA repair, recombination, transcription, telomere maintenance, and plays key roles in maintaining chromosome stability. The mutation of BLM helicase may lead to Bloom syndrome that characterized by genomic instability and a strong predisposition to many types of cancer. This study was studied the interaction of BLM helicase catalytic core （BLM^642 ～1290） with double-stranded DNA （dsDNA） by fluorescence polarization technology, and analyzed the related characteristic parameters to understand the DNA-binding and unwinding properties of BLM^642～1290 helicase. The results indicated that the binding and unwinding of the helicase and dsDNA was related to the length of 3＇-tailed single-stranded DNA （ssDNA） in dsDNA substrates; the helicase preferred to bind the ssDNA tails of dsDNA substrates, and every helicase molecule may bind 9.6 nt ssDNA; the unwinding efficiency was the highest level when the 3＇-tail ssDNA length of dsDNA substrates was 10nt, no longer increasing with the length. In addition, the blunt dsDNA was also bound and unwound by BLM^642～1290 helicase, but the binding affinity and the unwinding efficiency were smaller than 3＇-tailed dsDNA. It may be concluded that BLM^642～1290 helicase was monomer in binding and unwinding to dsDNA substrates, and may unwind dsDNA in an inchworm manner. These results will provide the theoretical foundation for further studying the unwinding mechanism of BLM helicase.

关 键 词：Bloom解旋酶 DNA结合 DNA解链 荧光偏振技术 

分 类 号：O657.32[理学—分析化学]