Cusick M E, Herman T M, DePamphilis M L, Wassarman P M
Biochemistry. 1981 Nov 10;20(23):6648-58. doi: 10.1021/bi00526a020.
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.
人们发现，正在复制的猴病毒40（SV40）染色体与其他真核染色体相似，即微球菌核酸酶（MNase）对其消化的速率和程度在正在复制的SV40染色质中比在非复制的成熟SV40染色质中更高。在完整细胞或核提取物中进行脉冲标记的正在复制的SV40染色体经MNase消化后，新生DNA迅速释放，形成双链DNA的基本裸露片段（3 - 7S），其平均长度为120个碱基对，并在反应过程中被降解。此外，还释放出了大小与成熟染色体中的核小体单体相当的核小体单体。另一方面，对均匀标记的成熟SV40染色体进行MNase消化，仅释放出核小体单体和寡聚体。从正在复制的染色体中释放出的小新生DNA片段代表了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，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'）对正在复制的SV40染色体进行预消化，导致PN - DNA完全降解。这一结果，连同分离的PN - DNA与SV40限制性片段的两条链退火效果相同的观察结果，表明PN - DNA起源于复制叉的两侧。超过90%的分离冈崎片段仅与逆向DNA模板退火。通过检测其对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中的浮力密度以及在琼脂糖凝胶上的大小，对分离出的PN - DNA的特性进行了评估。此外，还观察到纯化的SV40 DNA经MNase消化后也释放出一种大小与PN - DNA相似的瞬时中间体，这表明PN - DNA的出现不需要DNA - 蛋白质复合物。这些以及其他数据提供了一个正在复制的染色体模型，其中DNA合成发生在一个没有核小体的复制叉区域，该区域被指定为前核小体DNA。
