A binding site for Purα and Purβ is structurally unstable and is required for replication in vivo from the rat aldolase B origin

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• 作者：Yoshitaka Shimotai ; Hiroyuki Minami ; Yasushi Saitoh ; Yuichi Onodera ; Yukio Mishima ; Robert J. Kelm ; Jr. and Ken-ichi Tsutsumi
• 关键词：Mammalian replication origin ; Replicator ; Polypurine/polypyrimidine stretch ; Purα ; Purβ ; Aldolase B gene ; Chromatin immunoprecipitation
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2006
• 出版时间：10 February 2006
• 年：2006
• 卷：340
• 期：2
• 页码：517-525
• 全文大小：345 K

文摘

The rat aldolase B promoter acts as a replication origin in vivo, as well as an autonomously replicating sequence (ARS). Here, we examined roles of a polypurine stretch (site PPu) in this origin, which is indispensable to the ARS activity. Purification of site PPu-binding protein revealed that site PPu binds Purα and Purβ, i.e., single-stranded DNA-binding proteins whose roles in replication have been implicated, but less clear. Biochemical analyses showed that site PPu even in a longer DNA fragment is unstable in terms of double-helix, implying that Purα/β may stabilize single-stranded state. Deletion of site PPu from the origin DNA, which was ectopically positioned in the mouse chromosome, significantly reduced replicator activity. Chromatin immunoprecipitation experiments showed that deletion of site PPu abolishes binding of the Purα/β proteins to the origin. These observations suggest functional roles of site PPu and Purα/β proteins in replication initiation.