PURIFICATION OF RIP60 AND RIP100, MAMMALIAN PROTEINS WITH ORIGIN-SPECIFIC DNA-BINDING AND ATP-DEPENDENT DNA HELICASE ACTIVITIES

被引:64
作者
DAILEY, L
CADDLE, MS
HEINTZ, N
HEINTZ, NH
机构
[1] UNIV VERMONT,COLL MED,DEPT PATHOL,BURLINGTON,VT 05405
[2] ROCKEFELLER UNIV,MOLEC BIOL LAB,NEW YORK,NY 10021
[3] ROCKEFELLER UNIV,HOWARD HUGHES MED INST,NEW YORK,NY 10021
关键词
D O I
10.1128/MCB.10.12.6225
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Replication of the Chinese hamster dihydrofolate reductase gene (dhfr) initiates near a fragment of stably bent DNA that binds multiple cellular factors. Investigation of protein interactions with the dhfr bent DNA sequences revealed a novel nuclear protein that also binds to domain B of the yeast origin of replication, the autonomously replicating sequence ARS1. The origin-specific DNA-binding activity was purified 9,000-fold from HeLa cell nuclear extract in five chromatographic steps. Protein-DNA cross-linking experiments showed that a 60-kDa polypeptide, which we call RIP60, contained the origin-specific DNA-binding activity. Oligonucleotide displacement assays showed that highly purified fractions of RIP60 also contained an ATP-dependent DNA helicase activity. Covalent radiolabeling with ATP indicated that the DNA helicase activity resided in a 100-kDa polypeptide, RIP100. The cofractionation of an ATP-dependent DNA helicase with an origin-specific DNA-binding activity suggests that RIP60 and RIP100 may be involved in initiation of chromosomal DNA synthesis in mammalian cells.
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页码:6225 / 6235
页数:11
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