A binding site for Pur alpha and Pur beta is structurally unstable and is required for replication in vivo from the rat aldolase B origin.
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 Puralpha and Purbeta, 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 Puralpha/beta 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 Puralpha/beta proteins to the origin. These observations suggest functional roles of site PPu and Puralpha/beta proteins in replication initiation.
Shimotai
Yoshitaka
Y
Laboratory of Functional Genomics, United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan.
Minami
Hiroyuki
H
Saitoh
Yasushi
Y
Onodera
Yuichi
Y
Mishima
Yukio
Y
Kelm
Robert J
RJ
Tsutsumi
Ken-ichi
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D051381:Rats; D018741:Replication Origin / Q000235:genetics* / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C508559:PurA protein, rat; C508560:PurB protein, rat; D014157:Transcription Factors; D005634:Fructose-Bisphosphate Aldolase