EUKARYOTIC CHROMOSOME REPLICATION

复制是DNA的生物学作用的核心， 传承 DNA复制的控制确保染色体 以及时和精确的方式复制。 所有证据都表明 控制在复制开始时运行， 个体起源以高密度散布在真核染色体上。 我们打算研究的是复制起点的控制。 我们对这些控制的理解非常差，主要是因为 复制起点本身是难以捉摸的。 最近，两个敏感的 凝胶电泳技术已经被开发用于鉴定 染色体DNA 我们对酿酒酵母的研究 揭示了起源的起始活动在很大程度上取决于 他们的染色体背景。 语境控制的三个方面 将检查酵母中的来源使用。 (I)当原点位于 在质粒多聚体和rDNA基因座中， 不使用潜在的起源。 是什么导致了不活跃？ 是否存在间距限制？ 那么，如何选择有效的起源呢？ (II)转录活性rDNA重复序列中的复制起始 发生在非转录间隔区，复制是单向的， 移动转录方向的活动叉。 是否 其他染色体区域的转录活性影响起源使用？ 进化是否有利于允许复制的起源位置 分叉跟随转录复合体通过活跃转录的基因， 而不是和他们正面相撞 (III)染色体起源位于 端粒附近（染色体的物理末端）在S 相比起源位于内部网站。 端粒的特征是什么 影响起源激活时间，并且某些起源不活动， 它们与端粒的接近程度 我们处理这些问题的方法包括 在酵母质粒中进行定向替换和改变， 染色体，并使用我们最近开发的二维凝胶技术来识别 活动的起源，并估计其激活的效率。 的 这些问题的答案将导致更好地了解 调节正常细胞和那些 改变生长特性，如癌细胞。

Replication is central to the biological role of DNA as the molecule of inheritance. The control of DNA replication ensures that chromosomes are duplicated in a timely and precise way. All available evidence indicates that controls operate at the point of initiation of replication at individual origins scattered at high density over eukaryotic chromosomes. It is the control of replication origins that we propose to investigate. Our understanding of these controls is very poor, primarily because replication origins themselves have been elusive. Recently, two sensitive gel electrophesis techniques have been developed for identifying origins in chromosomal DNA. Our studies in the yeast Saccharomyces cerevisiae have revealed that the initiation activity of origins depends to a large extent on their chromosomal context. Three aspects of contextual control of origin use in yeast will be examined. (I) When origins are located in close tandem arrays--in plasmid multimers and in the rDNA locus--many potential origins are not used. What is responsible for the inactivity? Are there spacing constraints? And, how are the active origins chosen? (II) Replication initiation in the transcriptionally active rDNA repeats occurs in the non-transcribed spacer and replication is unidirectional with the active fork moving the transcriptional direction. Does the transcriptional activity of other chromosomal regions influence origin use? Has evolution favored the placement of origins that permits replication forks to follow transcription complexes through actively transcribed genes, rather than colliding head-on with them? (III) Chromosomal origins located near telomeres (the physical ends of chromosomes) are activated later in S phase than origins located at internal sites. What feature of telomeres influences origin activation times, and are some origins inactive because of their proximity to telomeres? Our approach to these questions involves making directed replacements and alterations in yeast plasmids and chromosomes and using our recently developed 2-D gel technique to identify active origins and to estimate the efficiency of their activation. The answers to these questions will lead to a greater understanding of the regulation of chromosome replication in normal cells and in those with altered growth properties, such as cancer cells.