DNA Helicases in Yeast DNA Replication and Repair

;​R o o t E n t y F qD @ C o m p o b j b W o r d d o c u m e n t O b j e c t P O O l ` 年代qD qDC：我想我是我的朋友F Microsoft Word 6.0文档MSWordDoc。6;​这些研究的一个工作模型是，在酵母染色体复制起点上，复制叉的组装发生在多个阶段：通过DNA聚合酶α引物酶进行DNA合成的识别、解绕、启动，以及pol δ和/或pol ε的募集。在一个复杂的、多组分的过程中，比如DNA复制，即使是对单个蛋白质最详尽的描述也不能描述实际生理事件的动力学。为了做到这一点，必须研究组件之间的相互作用，无论有多少组件。因此，该项目的实验主题是设计生化和遗传实验，以表征复制蛋白之间的动态相互作用，特别是解旋酶和形成原体和延伸装置的聚合酶。最终，我们希望用纯化的蛋白质在起源处重建一个解绕和起始事件。实验描述以DNA解旋酶为中心。特别是，P.I.通过筛选渗透细胞中DNA复制缺陷的突变体，发现了一种新的酵母DNA复制突变体dna2。提出的遗传和生化实验将测试Dna 2解旋酶参与酵母Dna复制的模式。特别是，解旋酶与三种必需的DNA聚合酶和必需的起源识别蛋白ORC的相互作用将被研究。有趣的是，Dna2解旋酶与一种特异性与HeLa MFl核酸酶相似的核酸酶结合，该酶参与SV40复制系统中滞后链DNA合成过程中Okazaki片段的成熟。酵母中有一种编码核酸酶的基因，叫做YKL510，具有类似的特异性，当它出现在高拷贝数的质粒上时，会抑制dna2突变。这表明YKL510在体外编码与Dna2p相关的核酸酶，而YKLS 10在体内可以与Dna2p相互作用。如果这些初步实验的解释是正确的，那么它们也指出了Dna2解旋酶在复制中的作用。该提案的第二部分将测试与Dna2相关的核酸酶是YKLS 10的假设，它们在体内确实存在关联，YKLS 10参与处理冈崎片段或酵母复制的其他方面。在过去的五年中，细胞生物学的研究发生了一场革命。分子方法在阐明细胞周期，即一个新细胞从亲本细胞产生的过程中是可能的。细胞周期的基本过程有两个；基因组的复制（DNA复制）和基因组分离到子细胞。这个建议的重点是DNA复制。私家侦探在这个过程中发现了一种新的，必需的酶。本提案中描述的复制酶（DNA解旋酶）的研究将增强我们对正常细胞如何将其DNA中包含的遗传信息从一代传递到下一代的理解。*** @ ....（）（）) ()() () Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT马西娅·斯坦伯格杰克·科恩@ S 5 @ M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M n2~ Microsoft Word 6.0e = e " " " " " " " L L L L L L L L n L @ C x | | | | | | |

; R o o t E n t r y F qD @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l `S qD `S qD C D E F G H I J K L F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; h.hB f s f M EU V v Ph Vh " I F Pj j` t v v Ph h.hBP F V V Ph " F F | t5 ~ u ~ t) D @ P B 7 F P D @ P " n F V ^ f M EU H f M 9507352 Campbell A working model for these studies is that assembly of a replication fork at a yeast chromosomal origin of replication occurs in multiple stages: recognition, unwinding, priming of DNA synthesis by DNA polymerase alpha primase, and recruitment of pol delta and/or pol epsilon. In a complicated, multicomponent process such as DNA replication, even the most exhaustive characterization of the individual proteins cannot describe the dynamics of the actual physiological events. In order to do that, interaction between the components, no matter how many there are, must be studied. The experimental theme of this project is therefore to design both biochemical and genetic experiments to characterize the dynamic interactions between the replication proteins, especially the helicases that unwind and the polymerases that form the primosome and elongation apparatus. Ultimately we would like to reconstitute an unwinding and initiation event at an origin with purified proteins. The experiments described center on DNA helicases. In particular, the P.I. has identified a new yeast DNA replication m utant, dna2, using a screen for mutants defective in DNA replication in permeabilized cells. The genetic and biochemical experiments proposed will test the mode of involvement of the Dna 2 helicase in yeast DNA replication. In particular, the interaction of the helicase with the three essential DNA polymerases and the essential origin recognition protein, ORC, will be studied. Interestingly, the Dna2 helicase copurifies with a nuclease whose specificity is similar to the HeLa MFl nuclease, that is involved in maturation of Okazaki pieces during lagging strand DNA synthesis in the SV40 replication system. A gene encoding a nuclease with similar specificity in yeast, called YKL510, suppresses the dna2 mutation when present on a high copy number plasmid. This suggests that YKL510 encodes the nuclease that is associated with Dna2p il vitro and that YKLS 10 can interact with Dna2p in vivo. If the interpretation of these preliminary experiments is correct, then they also point to a role for Dna2 helicase in replication. A second portion of the proposal will test the hypothesis that the nuclease associated with Dna2 is YKLS 10, that they do associate in vivo, and that YKLS 10 is involved in processing Okazaki pieces or some other aspect of replication in yeast. %% In the past five years there has been a revolution in the study of cell biology. Molecular approaches have been possible in elucidating the cell cycle, the process by which a new cell arises from a parent cell. Fundamental to the cell cycle are two processes; duplication of the genome (DNA replication) and segregation of the genome to daughter cells. This proposal focuses on DNA replication. The P.I. has discovered a new, essential enzyme for this process. Studies of the replication enzyme (the DNA helicase) described in this proposal will enhance our understanding of how normal cells transmit the genetic information contained in their DNA from one generation to the next. *** @ ....()()))()() Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg jack cohen @ S 5 @ M S u m m a r y I n f o r m a t i o n ( B 5 @ # qD @ 2~ Microsoft Word 6.0 4 e = e " " " " " " " L L L L L d n L @ C x | | | | | | |