DNA Helicases in Yeast DNA Replication and Repair

酵母 DNA 复制和修复中的 DNA 解旋酶

• 批准号: 9507352
• 负责人: Judith Campbell
• 金额: $ 24万
• 依托单位: California Institute of Technology
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9507352&HistoricalAwards=false
• 关键词: DNA; Helicases; Yeast; Replication; Repair

; 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 | | | | | | |