Lin S, Kowalski D
Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Mol Cell Biol. 1997 Sep;17(9):5473-84. doi: 10.1128/MCB.17.9.5473.
The DNA replication origins of the yeast Saccharomyces cerevisiae require several short functional elements, most of which are not conserved in sequence. To better characterize ARS305, a replicator from a chromosomal origin, we swapped functional DNA elements of ARS305 with defined elements of ARS1. ARS305 contains elements that are functionally exchangeable with ARS1 A and B1 elements, which are known to bind the origin recognition complex; however, the ARS1 A element differs in that it does not require a 3' box adjacent to the essential autonomously replicating sequence consensus. At the position corresponding to ARS1 B3, ARS305 has a novel element, B4, that can functionally substitute for every type of short element (B1, B2, and B3) in the B domain. Unexpectedly, the replacement of element B4 by ARS1 B3, which binds ABF1p and is known as a replication enhancer, inhibited ARS305 function. ARS305 has no short functional element at or near positions corresponding to the B2 elements in ARS1 and ARS307 but contains an easily unwound region whose functional importance was supported by a broad G+C-rich substitution mutation. Surprisingly, the easily unwound region can functionally substitute for the ARS1 B2 element, even though ARS1 B2 was found to possess a distinct DNA sequence requirement. The functionally conserved B2 element in ARS307 contains a known sequence requirement, and helical stability analysis of linker and minilinker mutations suggested that B2 also contains a DNA unwinding element (DUE). Our findings suggest that yeast replication origins employ a B2 element or a DUE to mediate a common function, DNA unwinding during initiation, although not necessarily through a common mechanism.
酿酒酵母的DNA复制起点需要几个短的功能元件,其中大多数元件在序列上并不保守。为了更好地表征来自染色体起点的复制子ARS305,我们将ARS305的功能DNA元件与ARS1的特定元件进行了交换。ARS305包含一些可与ARS1的A和B1元件进行功能交换的元件,已知这些元件可结合起点识别复合物;然而,ARS1的A元件不同之处在于它不需要紧邻基本自主复制序列共有序列的3'框。在与ARS1 B3对应的位置,ARS305有一个新元件B4,它可以在功能上替代B结构域中的每种短元件(B1、B2和B3)。出乎意料的是,用结合ABF1p且被称为复制增强子的ARS1 B3替换元件B4会抑制ARS305的功能。ARS305在与ARS1和ARS307中的B2元件对应的位置或其附近没有短功能元件,但包含一个易于解旋的区域,其功能重要性得到了广泛的富含G+C的取代突变的支持。令人惊讶的是,即使发现ARS1 B2具有独特的DNA序列要求,这个易于解旋的区域在功能上也可以替代ARS1 B2元件。ARS307中功能保守的B2元件包含一个已知的序列要求,对连接子和微型连接子突变的螺旋稳定性分析表明B2也包含一个DNA解旋元件(DUE)。我们的研究结果表明,酵母复制起点利用B2元件或DUE来介导一种共同功能,即起始过程中的DNA解旋,尽管不一定通过共同机制。