Lin S, Kowalski D
Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, NY 14263.
J Mol Biol. 1994 Jan 14;235(2):496-507. doi: 10.1006/jmbi.1994.1009.
Previous analysis of mutations in bacterial and yeast replication origins has identified a genetic component, termed a DNA unwinding element (DUE), whose intrinsic helical instability is essential for origin function. For the SV40 replication origin, we show here that the early palindrome (EP) and A + T-rich (AT) domains both exhibit helical instability, despite their dissimilar A + T compositions. To test the possible contribution of helical instability to SV40 origin function, the relationship between helical stability of mutant origin sequences and their known origin activity in vitro and in vivo was examined. Origin activity correlates inversely with the helical stability of mutations within the EP domain but not the AT domain or the T-antigen binding domain. The quantitative correlation holds for four different measures of origin activity determined in vitro and in vivo. An even better-correlated collection of mutations was found in a specific portion of the EP domain. This specific EP subdomain coincides with the sequence known to be strand-separated after T-antigen binds the origin in vitro and with the origin of bidirectional replication in vivo. Our analysis of origin mutations indicates that the helical instability of the specific EP subdomain is required to facilitate T-antigen-induced melting and the initiation of DNA replication. The sensitivity of the required EP subdomain to mutations that stabilize the DNA helix defines the DUE of the SV40 replication origin.
先前对细菌和酵母复制起点突变的分析已鉴定出一种遗传成分，称为DNA解旋元件（DUE），其固有的螺旋不稳定性对于起点功能至关重要。对于SV40复制起点，我们在此表明，早期回文（EP）和富含A + T（AT）的结构域均表现出螺旋不稳定性，尽管它们的A + T组成不同。为了测试螺旋不稳定性对SV40起点功能的可能贡献，研究了突变起点序列的螺旋稳定性与其在体外和体内已知的起点活性之间的关系。起点活性与EP结构域内突变的螺旋稳定性呈负相关，但与AT结构域或T抗原结合结构域无关。这种定量相关性适用于在体外和体内确定的四种不同的起点活性测量方法。在EP结构域的特定部分发现了一组相关性更好的突变。这个特定的EP亚结构域与已知在体外T抗原结合起点后发生链分离的序列以及体内双向复制起点一致。我们对起点突变的分析表明，特定EP亚结构域的螺旋不稳定性是促进T抗原诱导的解链和DNA复制起始所必需的。所需的EP亚结构域对稳定DNA螺旋的突变的敏感性定义了SV40复制起点的DUE。
