CIS-ACTING DETERMINANTS OF REPLICATION TIMING

癌症发生所需的细胞变化之一是基因组不稳定，这个术语描述了一系列导致基因突变频率增加和基因表达模式改变的途径。导致基因组不稳定的机制包括染色体断裂、基因扩增和基因表达水平的变化。后者通常与基因甲基化状态的变化有关。所有这些机制都与复制时间的变化相关，在某些情况下，复制时间的变化可能是这些现象的主要原因。到目前为止，很难评估复制计时变化在癌症发生中的作用，因为人们对控制复制计时的机制知之甚少。本申请中提出的实验旨在提供一个了解复制计时机制的切入点。我们打算利用我们实验室和其他实验室之前的工作，这些工作表明，在萌芽酵母中，ARS301和94bp的染色体DNA片段能够作为复制起点，但与以前研究的起点不同的是，它固有地复制较晚。同样令人感兴趣的是，ARS301是转录沉默元件的重要组成部分，转录抑制基因往往复制较晚。我们打算确定ARS301的延迟激发是由于存在晚复制决定序列，还是由于其中不存在早复制决定序列。在任何一种情况下，我们都将使用标准的突变技术，然后进行复制计时测试，以缩小并表征负责顺式作用的序列，以便它们可以用于帮助识别负责控制复制计时的DNA结合蛋白。

One of the cellular alterations required for development of cancer is genomic instability, a term that describes a collection of pathways all leading to increase frequency of gene mutation and altered patterns of gene expression. Among the mechanisms causing genomic instability are chromosome breakage, gene amplification, and changes in levels of gene expression. The latter are frequently associated with changes in gene methylation status. All of the mechanisms have been correlated with changes in replication timing, and it is possible that in some cases changes in replication timing may be the primary cause of these phenomena. So far it has been difficult to evaluate the role of changes in replication timing in cancer development, because so little is known about the mechanisms controlling replication timing. The experiments proposed in this application are intended to provide an entry point into understanding the mechanism of replication timing. We intend to take advantage of previous work in our laboratory and others which shows that ARS301, and 94-bp stretch of chromosomal DNA in the budding yeast, Saccharomyces cerevisiae, is capable of serving as a replication origin but differs from previously studied origins in being inherently late replicating. It is also of interest that ARS301 is an important part of a transcriptional silencer element, and transcriptionally repressed genes tend to replicate late. We intend to determine whether the late firing of ARS301 is due to the presence of late-replication-determining sequences or to the absence of early-replication determining sequences within it. In either case, we shall employ standard mutagenic techniques followed by replication timing tests to narrow down and characterize the responsible cis-acting sequences, so that they can be used to help identify the DNA-binding proteins that are responsible for controlling replication timing.