Mastrangelo I A, Held P G, Dailey L, Wall J S, Hough P V, Heintz N, Heintz N H
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
J Mol Biol. 1993 Aug 5;232(3):766-78. doi: 10.1006/jmbi.1993.1430.
We show assembly of low and high multimers of HeLa cell nuclear protein, RIP60, at the origin of bidirectional replication (OBR) identified by Burhans, Vassilev, Caddle, Heintz and DePamphilis in Chinese hamster ovary cells. RIP60 binds a 5'-ATT-3' reiterated sequence downstream of the OBR and a second, homologous ATT sequence of opposite orientation situated within the OBR zone. Specifically bound structures were studied by conventional electron microscopy (EM) and quantitative scanning transmission electron microscopy (STEM). Dimers and multiples of dimers link the downstream binding site that overlaps a bent DNA sequence and the homologous upstream OBR sequence, looping out 700 bp of intervening DNA. Superposed dimers are found at individual unlinked sites, stabilized presumably through protein-protein interaction, and such superposition appears to occur also in the basic link structure. Along the loop, single crossovers and extended twists are observed by conventional EM. By STEM, loop DNA is laterally compacted, with diameter and mass equivalent to double-duplex DNA strands. Supercoiled 736 bp and 5243 bp circular DNAs assume similar laterally compacted geometries that are mostly absent from relaxed forms. These observations parallel the compacted, interwound superhelices viewed by cryo-electron microscopy in vitrified solutions containing magnesium ions, and provide structural evidence in agreement with that from conventional EM for superhelical tension in RIP60 loop DNA. Loop superhelicity could arise as a topological response to linking and suggests a functional role for link formation.
我们展示了HeLa细胞核蛋白RIP60的低聚物和高聚物在中国仓鼠卵巢细胞中由Burhans、Vassilev、Caddle、Heintz和DePamphilis鉴定出的双向复制起点（OBR）处的组装情况。RIP60结合在OBR下游的一个5'-ATT-3'重复序列以及位于OBR区域内的另一个反向同源ATT序列。通过传统电子显微镜（EM）和定量扫描透射电子显微镜（STEM）研究了特异性结合结构。二聚体和二聚体的倍数连接了与弯曲DNA序列重叠的下游结合位点和同源的上游OBR序列，使700 bp的中间DNA形成环。在单个未连接的位点发现了叠加的二聚体，推测是通过蛋白质-蛋白质相互作用稳定的，并且这种叠加似乎也发生在基本的连接结构中。通过传统EM观察到沿着环有单交叉和延伸的扭曲。通过STEM观察到，环DNA在横向被压缩，其直径和质量与双链DNA链相当。超螺旋的736 bp和5243 bp环状DNA呈现出相似的横向压缩几何形状，而松弛形式中大多不存在这种形状。这些观察结果与在含有镁离子的玻璃化溶液中通过冷冻电子显微镜观察到的压缩、相互缠绕的超螺旋一致，并为RIP60环DNA中的超螺旋张力提供了与传统EM一致的结构证据。环超螺旋可能作为对连接的拓扑响应而出现，并暗示了连接形成的功能作用。
