自《线粒体DNA：研究方法与实验方案（原著第2版）（导读版）》第1版以来，被报道与生物能量代谢异常、细胞死亡及疾病相关的mtDNA突变在不断增加。同样mtDNA体细胞突变的生物学功能的了解也在继续提高。在第2版中，来自8个国家的专家分析了他们在mtDAN研究领域各芳名的专业知识和详细的研究方法。《线粒体DNA：研究方法与实验方案（原著第2版）（导读版）》分为三部分，涵盖了从mtDNA分子到氧化呼吸链复合物合成组装这一通路的相关研究方法、线粒体活性氧簇（ROS）长生了mtDNA损伤和修复、mtDNA异质性突变的鉴定和定量等内容。

      《线粒体DNA：研究方法与实验方案（原著第2版）（导读版）》秉承Springer《分子生物学方法》系列丛书的一贯风格，阐述明晰、便于使用，每章包括对相关问题的介绍，所需材料和试剂的清单，实验操作的具体步骤，以及常见问题的解决方法和缺陷规避。

前言
撰稿人
第一部分:线粒体DNA复制、转录、翻译和呼吸链复合物组装
1.动物细胞mtDNA核状小体的生化提取 Daniel F.Bogenhangen
2.二维琼脂糖凝胶电泳分析线粒体DNA Aurelio Reyes,Takehior Yasukawa,Tricia J.Cluett,and Ian J.Holt
3.果蝇和人线粒体DNA复制蛋白:DNA聚合酶r和线粒体单链DNA结合蛋白的比较纯化策略 Marcos T.Oliweira and Lautie S.Kaguni
4.参与人类疾病的线粒体DNA聚合酶突变体的功能分析 Sherine S.L.Chan and Willianm C.Copeland
5.人类线粒体单链DNA结合蛋白的纯化制备 Matthew J.Longley,Leslie A.S.mith,ang Willian C.Copeland
6.评估线粒体转录因子A(TFAM)与DNA结合的方法 Atsushi Fukuoh and Dongchon Kang
7.线粒体DNA解旋酶在人类细胞中的诱导表达、纯化和体外功能分析 Steffi Goffart and Hans Spelbrink
8.人类线粒体DNA解旋酶重组蛋白的纯化策略 Tawn D.Ziebarth and Laurie S.Kaguni
9.用提取的蛋白组分研究线粒体转录终止的方法 Paola Loguercio Polosa,Stefania Deceglie,Marian Roberti,Maria Nicola Gadaleta,and Palmito Cantatore
10.氧化磷酸化:线粒体编码蛋白的合成以及将单独的结构亚基组装成具有功能的全酶复合物 Scot C.Leary and Florin Sasarman
第二部分:线粒体DNA的损伤和修复
11.线粒体活性氧簇的生成 Adrian J.Lambert and Martin D.Brand 前言
撰稿人
第一部分:线粒体DNA复制、转录、翻译和呼吸链复合物组装
1.动物细胞mtDNA核状小体的生化提取 Daniel F.Bogenhangen
2.二维琼脂糖凝胶电泳分析线粒体DNA Aurelio Reyes,Takehior Yasukawa,Tricia J.Cluett,and Ian J.Holt
3.果蝇和人线粒体DNA复制蛋白:DNA聚合酶r和线粒体单链DNA结合蛋白的比较纯化策略 Marcos T.Oliweira and Lautie S.Kaguni
4.参与人类疾病的线粒体DNA聚合酶突变体的功能分析 Sherine S.L.Chan and Willianm C.Copeland
5.人类线粒体单链DNA结合蛋白的纯化制备 Matthew J.Longley,Leslie A.S.mith,ang Willian C.Copeland
6.评估线粒体转录因子A(TFAM)与DNA结合的方法 Atsushi Fukuoh and Dongchon Kang
7.线粒体DNA解旋酶在人类细胞中的诱导表达、纯化和体外功能分析 Steffi Goffart and Hans Spelbrink
8.人类线粒体DNA解旋酶重组蛋白的纯化策略 Tawn D.Ziebarth and Laurie S.Kaguni
9.用提取的蛋白组分研究线粒体转录终止的方法 Paola Loguercio Polosa,Stefania Deceglie,Marian Roberti,Maria Nicola Gadaleta,and Palmito Cantatore
10.氧化磷酸化:线粒体编码蛋白的合成以及将单独的结构亚基组装成具有功能的全酶复合物 Scot C.Leary and Florin Sasarman
第二部分:线粒体DNA的损伤和修复
11.线粒体活性氧簇的生成 Adrian J.Lambert and Martin D.Brand
12.超螺旋敏感的qPCR方法检测mtDNA损伤 Sam W.Chan and Junjian Z.Chen
13.免疫荧光法定量分析线粒体DNA中氧化形式的鸟瞟岭(8-起鸟瞟岭) Mizuki Ohno,Sugako Oka,and Yusaku Nakabeppu
14.在分离的线粒体中体外检测DNA碱基切除修复能力 Melissa M.Page and Jeffrey A. Stuart
15.利用稳定转染、瞬时转染、病毒转导及TAT-介导的蛋白转导方法靶向哺乳动物细胞线粒体导入修复蛋白 Christopher A.Koczor,Janet W.Snyder,Inna N. Shokolenko,Allison W.Dobson,Glenn L.Wilson,and Susan P.LeDoux
16.一种局限线粒体DNA而非核DNA的氧化损伤修复缺陷的细胞系的构建和功能分析 Sugako Oka,Mizuki Ohno,and Yusaku Nakabeppu
第三部分:线粒体DNA突变
17.酿酒酵母中线粒体DNA的氧化损伤和突变诱变 Lyra M.Griffiths,Nicole A.Doudican,Doudican,Gerald S.Shadel,and Paul W.Doetsch
18.基于变性高效液相色谱(DHPLC)异源双链分析法测定人类组织中DNA突变异质性 Kok Seong Lim,Robert K.Naviaux,and Richard H.Haas
19.利用特异识别异源双链错配位点的Surv or核酸酶快速检测未知的异质性线粒体DNA突变 Sylvie Bannnwarth,Vincent Procaccio,and Véronique Paquis-Flucklinger
20.用于单细胞线粒体DNA突变分析的细胞分离和收集方法 Yevgenya Kraytsberg,Natalya Bodyak,Susan Myerow,Alexander Nicholas,Konstantin Ebralidze,and Konstantin Khrapko
21.采用单细胞单分子PCR定量分析线粒体DNA体细胞突变 Yevgenya Kraytsberg,Natalya Bodyak,Susan Myerow,Alexander Nicholas,Konstantin Ebralidze,and Konstantin Khrapko
22.线粒体中DNA前体库(dNTP库)的测定 Christopher K.Mathews and Linda J.Wheeler
23.构建缺乏线粒体DNA的人类细胞系 Kazunari Hashiguchi and Qiu-Mei Zhang-Akiyama
索引

Chapter 1
Biochemical Isolation of mtDNA Nucleoids from Animal Cells
Daniel F. Bogenhagen
Abstract
Mitochondrial DNA (mtDNA) in animal cells is organized into clusters of 5–7 genomes referred to as nucleoids. Contrary to the notion that mtDNA is largely free of bound proteins, these structures are nearly as rich in protein as nuclear chromatin. While the purification of intact, membrane-bound mitochondria is an established method, relatively few studies have attempted biochemical purification of mtDNA nucleoids. In this chapter, two alternative methods are presented for the purification of nucleoids. The first method yields the so-called native nucleoids, using conditions designed to preserve non-covalent protein–DNA and protein–protein interactions. The second method uses formaldehyde to crosslink proteins to mtDNA and exposes nucleoids to treatment with harsh detergents and high salt concentrations.
Key words: mtDNA, mitochondria, nucleoids, chromatin IP.
1. Introduction
The maintenance of mitochondria depends on the mitochondrial DNA (mtDNA) for synthesis of several protein components of the oxidative phosphorylation machinery. In mammals, 13 proteins are encoded in the mtDNA genome along with 12S and 16S rRNAs and a complete, albeit minimal, complement of 22 tRNAs. The 13 proteins synthesized on mitochondrial ribosomes are incorporated into respiratory complexes I, III, IV, and V along with approxi-mately 67 nucleus-encoded subunits (1). Cells typically maintain thousands of copies of mtDNA distributed among hundreds of organelles that exchange components through active cycles of fusion and fission (2, 3). These mtDNA genomes are organized in nucleoids containing 2–10 genomes, as indicated in Table 1.1 (4).
Jeffrey A. Stuart (ed.), Mitochondrial DNA, Methods and Protocols, vol. 554 aHumana Press, a part of Springer Science+Business Media, LLC 2009 DOI 10.1007/978-1-59745-521-3_1 Springerprotocols.com
Bogenhagen
Table 1.1 Number of nucleoids and mtDNA molecules per nucleoid in various cell types
Cell type Nucleoids/cell mtDNA/cell mtDNA/focus Reference
Using gold-labeled anti-DNA antibodies, Iborra et al. (5) have estimated that mtDNA nucleoids have an average diameter of 70 nm, indicating that several mtDNA genomes are very tightly packaged. CsCl density gradient analysis of crosslinked mtDNA nucleoid preparations yields a density of approximately 1.5 g/ml, consistent with an approximately equal content of protein and DNA. Thus, a nucleoid with 7 mtDNA genomes may be expected to have a mass of about 140 MDa, half of which is protein.
Time-lapse imaging has shown that nucleoids in mammalian cells are relatively stable structures rather evenly spaced within the mitochondrial reticulum (6, 7). This has encouraged several laboratories to try to biochemically isolate nucleoids to identify proteins associated with the mtDNA genome. Such studies have routinely identified two well-established mtDNA-binding pro-teins as nucleoid markers. These are the mitochondrial single-stranded DNA-binding protein, mtSSB (8), a tetramer of 16 kDa subunits homologous to Escherichia coli SSB, and TFAM, an abundant HMG-box protein that binds duplex DNA with limited sequence specificity. Kang’s group has estimated that HeLa cells contain as many as 3,000 copies of mtSSB and 1,700 copies of TFAM per mtDNA molecule (9). However, other groups have suggested that the TFAM content is considerably lower (10). Both of these proteins occur with a pool of free proteins in equilibrium with the fraction bound to mtDNA. This is likely to be true for other proteins involved in mtDNA replica-tion and transcription as well. For any individual nucleoid-associatedprotein,thefractionofproteinretainedinabiochemical preparation of nucleoids is expected to vary depending on the conditionsusedinthefractionationprocedure.Investigatorsinter-ested in studying mtDNA nucleoid proteins must be cognizant of this limitation.
A wide variety of proteins have been found in association with mtDNA nucleoids isolated from either yeast or animal cell sources. The list includes several proteins with known roles in mtDNA replication and transcription, which may be considered ‘‘positive controls,’’ along with a number of chaperones and metabolic
Purification of Native and Crosslinked mtDNA Nucleoids
proteins. Other reviews have summarized the results of such studies; it is not the goal of this methods’ chapter to recapitulate or interpret these results. Instead, this chapter will present two alternative procedures for biochemical preparation of nucleoids. Kaufman et al. (11) have previously presented a protocol for for-maldehyde crosslinking of proteins to mtDNA in this series. A comparison of the nucleoid protein composition obtained using gentle handling of native (non-crosslinked) complexes with that obtained using formaldehyde crosslinking coupled with harsh solution conditions provides significant insight into the structure of nucleoids. The results of this comparison suggest that nucleoids can be considered to contain a core of tightly associated proteins, including TFAM, mtSSB, mitochondrial DNA and RNA poly-merases, surrounded by additional metabolic proteins and chaper-ones.Atthisjuncture,itisnotasimplemattertodecideifaprotein contained in a biochemical preparation enriched in nucleoids is an ‘‘authentic’’ nucleoid protein. It is hoped that these methods will contribute to further efforts to identify and characterize proteins important for the maintenance and expression of mtDNA.
Both methods described below begin with the preparation of highly purified mitochondria based on a protocol presented pre-viouslyinthisseries (2).Thisprotocolisoneofthemanyavailablein the literature, and it is not possible to review it in detail in the space provided. Methods for mitochondrial purification typically take advantage of the membrane-delimited nature of the organelle and its distinctive size and density. Following cell disruption, mitochon-dria must be handled in isotonic buffers like the mannitol–sucrose buffer described below to avoid organelle rupture. Detergents must be rigorously avoided during mitochondrial purification, although differential extraction with digitonin is often used to prepare mito-plastslackingtheoutermembrane.Insomecases,mitochondriacan be purified by differential sedimentation, particularly if the source is enriched in mitochondria. However, it is always desirable to use a purification procedure that employs centrifugation in a gradient containing sucrose or other separation media in order to remove contaminating organelles of both higher and lower densities. Finally, it is critically important to recognize that even density gradient purified mitochondria are routinely contaminated with associated nuclear DNA. This contamination is well known to researchers with experience in biochemical isolation of mtDNA as ethidium bromide–CsCl gradients routinely show a heavy upper band of nuclear DNA (2). Nuclear DNA contamination can be reduced,butnoteliminated,bynucleasetreatmentofmitochondria at an intermediate stage in purification, as shown in the agarose gel analysis in (Fig. 1.1). The undigested DNA in a typical HeLa mitochondrial preparation contains a broad zone of high molecular weight DNA that appears to run as a band since it has been sheared by pipetting to a relatively uniform size of about 20–30 kb. When
Bogenhagen
the DNA is digested with restriction endonuclease HindIII, the expected mtDNA fragments of 10.2 and 5.5 kb are resolved over a smear of nuclear DNA fragments. Treatment of mitochondria with DNase I at an intermediate step in purification reduces this back-ground smear. However, low molecular weight fragments of nuclear DNA are retained in the mitochondrial fraction (not shown in Fig. 1.1). If the extent of nuclease digestion is sufficient, most of these fragments will be removed by a stringent sizing step that takes advantage of the large size of mtDNA nucleoids. This DNA contamination can introduce some non-mitochondrial pro-teins, such as histones, into nucleoid preparations.
2. Materials
2.1. MSH and Nuclease Treatment Buffer
Both buffers include protease inhibitors at final concentrations of 5 mg/ml leupeptin, 2 mg/ml E64, 1 mM pepstatin, and 0.2 mM PMSF.
1. MSH: 210 mM mannitol, 70 mM sucrose, 20 mM HEPES, pH 8, 2 mM EDTA, 2 mM DTT
2.2. Anti-TFAM and Anti-mtSSB Antibody Columns for Affinity Purification
2.3. Solutions for Sedimentation of Formaldehyde Crosslinked Nucleoids
Purification of Native and Crosslinked mtDNA Nucleoids