MBP基于标记策略的Mgm101重组蛋白的制备
Summary
酵母线粒体类核蛋白质，Mgm101，是一种重组蛋白RAD52型，形成大低聚环。甲协议的描述以制备可溶性的重组Mgm101使用麦芽糖结合蛋白（MBP）标记加上阳离子交换和尺寸排阻色谱法策略。
Abstract
20年前被确定MGM101基因维护线粒体DNA中的作用。从几组研究表明，的Mgm101蛋白参与线粒体DNA的重组修复。最近的调查表明，Mgm101 RAD52型重组蛋白家族有关。这些蛋白质形成大的低聚环促进退火同源单链DNA分子。然而，表征阻碍了Mgm101的生产重组蛋白的难度。在这里，用于制备重组Mgm101的一个可靠的程序进行说明。麦芽糖结合蛋白（MBP）标签Mgm101的首先在大肠杆菌中表达。最初是由直链淀粉的亲和层析柱纯化的融合蛋白。在被释放后通过蛋白水解裂解，Mgm101从MBP分离，通过阳离子交换色谱法。然后单分散Mgm101是获得通过尺寸排阻色谱法。可以定期获得的产量为〜0.87毫克每升细菌培养Mgm101。该的重组Mgm101有DNA污染最小。对样品成功地用于生化，结构和单粒子图像分析Mgm101。此步骤也可用于制备其它大的低聚物的DNA结合蛋白，可能会被错误折叠的和细菌细胞毒性的。
Introduction
同源重组是非常重要的双链断裂的修复（DNA双链断裂）和链间交联，并从倒塌的复制叉1重新开始DNA复制。在常规的同源重组，中央的反应由ATP-依赖的重组酶RecA蛋白在原核细胞中，在真核生物中1-3的Rad51和Dmc1。这些重组酶形成核蛋白丝的单链DNA，这是必不可少的双链DNA模板（ 图1，左侧面板）4-7内发起同源搜索和钢绞线入侵。除了 常规的计划，同源重组也可以采取地方在RecA/Rad51-independent的方式（ 图1，右图）。例如，酵母的保守和Rad59蛋白质可以直接催化切除双链DNA断裂露出的互补单链DNA链退火。这个重组过程中，被称为唱乐链退火，一般不涉及同源配对的双链DNA模板。退火后，除去核酸外切酶的异源尾巴刻痕结扎以恢复基因组连续性8-10。常伴有直接重复区域之间的基因组序列缺失修复由单链退火机制。
RAD52属于普遍噬菌体11的重组蛋白质，是一个多元化的群体。这些蛋白质也被称为单链退火蛋白（会计实务准则），根据他们的活动在促进同源的单链DNA分子退火。最好噬菌体会计实务准则Redβ的的ERF从噬菌体λ噬菌体rac和葡激酶蛋白从噬菌体lactococcal UL36和P22，RECT。的会计准则结构其特征在于由一个典型的β-β-β-α倍，虽然相似，几乎检测不到能够在他们的主要序列。他们都形成大的同源寡聚体环10 – 14倍的对称性在体外 12-14。这一特点高阶的组织结构功能的影响还不是很清楚。
我们有兴趣了解线粒体基因组同源重组的机制。我们先前已经确定MGM101是必不可少的维护在酿酒酵母 15线粒体DNA的基因。MGM101后来发现线粒体拟核相关需要的耐受性线粒体DNA损伤剂16。然而，这项研究已Mgm101忍住在过去十年中的难度生产重组Mgm101。最近，我们已经成功地大量生产可溶性Mgm101在从E大肠杆菌使用的MBP融合策略。这使我们能够证明Mgm101股生化和结构的相似性与RAD52家族蛋白17,18。在这份报告中，三个步骤的纯化过程进行了描述，产生均匀Mgm101for的生化和结构分析（ 图2）。
Protocol
Representative Results
Discussion
它一直是一个挑战，产生一个稳定的，原生的重组Mgm101蛋白E.大肠杆菌可能是由于其在细菌细胞中的不溶性。在这份报告中，我们表明，MBP融合战略使蛋白质表示在相当高的水平。通过负染色透射电子显微镜和尺寸排阻色谱法，我们以前曾表明，MBP的融合蛋白在体外 18形成均匀的低聚物。这是可能的折叠和低聚Mgm101的，可能比较慢线粒体基质相比，细菌细胞中。 unoligomerized Mg…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢斯蒂芬·威尔肯斯在透射电子显微镜的帮助。这项工作是支持由国家机构卫生部授予R01AG023731的。
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| Expression vector pMAL-c2E | New England Biolabs | #N8066S | |
| PreScission Protease | GE Healthcare Life Sciences | #27-0843-01 | |
| BL21-CodonPlus(DE3)-RIL cells | Strategene | #230245 | |
| Leupeptin | Roche Applied Science | #11034626001 | |
| Pepstatin | Roche Applied Science | #11359053001 | |
| Phenylmethylsulfonyl fluoride (PMSF) | Roche Applied Science | #10837091001 | |
| DNAse I | Sigma | #DN25-1G | |
| Isopropyl β-D-1-thiogalactopyranoside (IPTG) | Roche Applied Science | #11411446001 | |
| Amylose resin | New England Biolabs | #E8021L | |
| Econo-Column chromatography column | BIO-RAD | #7372512 | |
| Bio-Scale Mini Macro-Prep High S cartridge (1 ml) | BIO-RAD | #732-4130 | |
| VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO) | Sartorius Stedium | #VS15RH02 | |
| Superose 6 prep grade column | Amersham Bioscirnces | #17-0489-01 | |
| VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO) | Sartorius Stedium | #VS0611 |
Referenzen
- San Filippo, J., Sung, P., Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229-257 (2008).
- Bishop, D. K., Park, D., Xu, L., Kleckner, N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell. 69, 439-456 (1992).
- Shinohara, A., Ogawa, H., Ogawa, T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 69, 457-470 (1992).
- Passy, S. I., et al. Human Dmc1 protein binds DNA as an octameric ring. Proc. Natl. Acad. Sci. U.S.A. 96, 10684-10688 (1999).
- Story, R. M., Weber, I. T., Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature. 355, 318-325 (1992).
- Yu, X., Jacobs, S. A., West, S. C., Ogawa, T., Egelman, E. H. Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA. Proc. Natl. Acad. Sci. U.S.A. 98, 8419-8424 (2001).
- Conway, A. B., et al. Crystal structure of a Rad51 filament. Nat. Struct. Mol. Biol. 11, 791-796 (2004).
- Bai, Y., Davis, A. P., Symington, L. S. A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59. Genetik. 153, 1117-1130 (1999).
- Bai, Y., Symington, L. S. A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 10, 2025-2037 (1996).
- Paques, F., Haber, J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349-404 (1999).
- Lopes, A., Amarir-Bouhram, J., Faure, G., Petit, M. A., Guerois, R. Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs. Nucleic Acids Res. 38, 3952-3962 (2010).
- Poteete, A. R., Sauer, R. T., Hendrix, R. W. Domain structure and quaternary organization of the bacteriophage P22 Erf protein. J. Mol. Biol. 171, 401-418 (1983).
- Passy, S. I., Yu, X., Li, Z., Radding, C. M., Egelman, E. H. Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins. Proc. Natl. Acad. Sci. U.S.A. 96, 4279-4284 (1999).
- Ploquin, M., et al. Functional and structural basis for a bacteriophage homolog of human RAD52. Curr. Biol. 18, 1142-1146 (2008).
- Chen, X. J., Guan, M. X., Clark-Walker, G. D. MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae. Nucl. Acids Res. 21, 3473-3477 (1993).
- Meeusen, S., et al. Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA. J. Cell Biol. 145, 291-304 (1999).
- Mbantenkhu, M., et al. Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination. J. Biol. Chem. 286, 42360-42370 (2011).
- Nardozzi, J. D., Wang, X., Mbantenkhu, M., Wilkens, S., Chen, X. J. A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein. Mgm101. J. Biol. Chem. 287, 37259-37268 (2012).
- Zuo, X., Xue, D., Li, N., Clark-Walker, G. D. A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele. FEMS Yeast Res. 7, 131-140 (2007).
- Itoh, K., et al. DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum. Mitochondrion. 11, 575-586 (2011).
- Janicka, S., et al. A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination. Plant J. 72, 423-435 (2012).
