FORMALDEHYDE-MEDIATED DNA PROTEIN CROSSLINKING - A PROBE FOR INVIVO CHROMATIN STRUCTURES
FORMALDEHYDE-MEDIATED DNA PROTEIN CROSSLINKING - A PROBE FOR INVIVO CHROMATIN STRUCTURES
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DOI：
10.1073/pnas.82.19.6470
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
VARSHAVSKY, A
中科院分区：
文献类型：
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作者：
SOLOMON, MJ;VARSHAVSKY, A
甲醛(HCHO)在体外和体内都能产生DNA-蛋白质交联物。猴病毒40(SV40)染色体在体外或体内(在SV40感染的细胞内)与HCHO长时间孵育固定后，在NaDodSO4存在下，通过Pronase的限制消化可以转化为几乎不含蛋白质的DNA。剩余的抗Pronase的DNA多肽加合物在凝胶电泳法上延迟DNA，允许分离游离和含有交联键的DNA。尽管在体外和体内都能有效地将组蛋白与核小体内的DNA交联，但在体外，HCHO不会将纯化的lac抑制物与含有lac操纵子的DNA或(A+T)-DNA结合蛋白(α-蛋白)与其同源DNA交联。此外，一种不与DNA结合的蛋白质，如血清白蛋白，即使在极高的蛋白质浓度下也不会通过HCHO与DNA交联。甲醛作为DNA-蛋白质交联剂的这些性质被用来探测核小体在体内的分布。我们发现，在体内的SV40染色体的子集中，跨越SV40染色体的“暴露”(核酸酶敏感)区域的325个碱基对的伸展范围内，没有可与HCHO交联的DNA-蛋白质接触。这一复制起始-近端区域先前已被发现在分离的SV40染色体的子集中缺乏核小体。我们讨论了HCHO技术的其他应用，包括在活体核小体“足迹”中获得碱基分辨率的可能性。
Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO4. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (.alpha.-protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. We show that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the "exposed" (nuclease-hypersensitive) region of the SV40 chromosome. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. We discuss other applications of the HCHO technique, including the possibility of obtaining base-resolution in vivo nucleosome "footprints.".