Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease.
Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease.
复制标题
脱氧核糖核酸复制叉处的染色质结构：核小体前的脱氧核糖核酸被微球菌核酸酶从复制的猿猴病毒 40 号染色体上快速切除。
DOI：
10.1021/bi00526a020
复制
发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
P. M. Wassarman
中科院分区：
文献类型：
--
作者：
M. Cusick;T. Herman;M. DePamphilis;P. M. Wassarman
复制猿猴病毒40（SV 40）染色体被发现是类似于其他真核细胞染色体的微球菌核酸酶（MNase）消化的速度和程度是更大的复制比非复制成熟SV 40染色质。在完整细胞或核提取物中脉冲标记的复制的SV 40染色体的MNase消化导致新生DNA作为双链体DNA（3-7S）的基本上裸露的片段快速释放，所述双链体DNA（3-7S）具有120个碱基对的平均长度并且在反应过程中降解。此外，释放出大小与成熟染色体相当的核小体单体。另一方面，MNase消化均匀标记的成熟SV 40染色体导致仅释放核小体单体和寡聚体。从复制染色体释放的新生DNA小片段代表来自复制叉区域的前核小体DNA（PN-DNA），该区域包含DNA合成的实际位点，并包括冈崎片段。用大肠杆菌核酸外切酶III（3 '-5'）和噬菌体T7基因6核酸外切酶（5 '-3'）预消化复制的SV 40染色体，导致PN-DNA完全降解。这一结果，以及观察到分离的PN-DNA与SV 40限制性片段的两条链同样良好地退火，证明PN-DNA来源于复制叉的两侧。超过90%的孤立冈崎片段退火只逆行DNA模板。通过检测PN-DNA对MNase和单链特异性S1核酸内切酶的敏感性、脱蛋白前后的沉降行为、甲醛处理后在CsCl中的浮力密度以及琼脂糖凝胶上的大小来评估分离的PN-DNA的特性。此外，观察到纯化的SV 40 DNA的MNase消化也导致释放大小与PN-DNA相似的瞬时中间体，表明不需要DNA-蛋白质复合物来解释PN-DNA的出现。这些和其他数据提供了一个复制染色体的模型，其中DNA合成发生在复制叉区域，该区域不含核小体，并被称为前核小体DNA。
Replicating simian virus 40 (SV40) chromosomes were found to be similar to other eukaryotic chromosomes in that the rate and extent of micrococcal nuclease (MNase) digestion were greater with replicating than with nonreplicating mature SV40 chromatin. MNase digestion of replicating SV40 chromosomes, pulse labeled in either intact cells or nuclear extracts, resulted in the rapid release of nascent DNA as essentially bare fragments of duplex DNA (3-7S) that had an average length of 120 base pairs and were degraded during the course of the reaction. In addition, nucleosomal monomers, equivalent in size to those from mature chromosomes, were released. On the other hand, MNase digestion of uniformly labeled mature SV40 chromosomes resulted in the release of only nucleosomal monomers and oligomers. The small nascent DNA fragments released from replicating chromosomes represented prenucleosomal DNA (PN-DNA) from the region of replication forks that encompasses the actual sites of DNA synthesis and includes Okazaki fragments. Predigestion of replicating SV40 chromosomes with both Escherichia coli exonuclease III (3'-5') and bacteriophage T7 gene 6 exonuclease (5'-3') resulted in complete degradation of PN-DNA. This result, together with the observation that isolated PN-DNA annealed equally well to both strands of SV40 restriction fragments, demonstrated that PN-DNA originates from both sides of replication forks. Over 90% of isolated Okazaki fragments annealed only to the retrograde DNA template. The characteristics of isolated PN-DNA were assessed by examining its sensitivity to MNase and single strand specific S1 endonuclease, sedimentation behavior before and after deproteinization, buoyant density in CsCl after formaldehyde treatment, and size on agarose gels. In addition, it was observed that MNase digestion of purified SV40 DNA also resulted in the release of a transient intermediate similar in size to PN-DNA, indicating that a DNA-protein complex is not required to account for the appearance of PN-DNA. These and other data provide a model of replicating chromosomes in which DNA synthesis occurs on a region of replication forks that is free of nucleosomes and is designated as prenucleosomal DNA.