Binding of a sequence-specific single-stranded DNA-binding factor to the simian virus 40 core origin inverted repeat domain is cell cycle regulated.
The inverted repeat domain (IR domain) within the simian virus 40 origin of replication is the site of initial DNA melting prior to the onset of DNA synthesis. The domain had previously been shown to be bound by a cellular factor in response to DNA damage. We demonstrate that two distinct cellular components bind opposite strands of the IR domain. Replication protein A (RPA), previously identified as a single-stranded DNA binding protein required for origin-specific DNA replication in vitro, is shown to have a preference for the pyrimidine-rich strand. A newly described component, IR factor B (IRF-B), specifically recognizes the opposite strand. IRF-B binding activity in nuclear extract varies significantly with cell proliferation and the cell cycle, so that binding of IRF-B to the IR domain is negatively correlated with the onset of DNA synthesis. Loss of IRF-B binding from the nucleus also occurs in response to cellular DNA damage. UV cross-linking indicates that the core binding component of IRF-B is a protein of ca. 34 kDa. We propose that RPA and IRF-B bind opposite strands of the IR domain and together may function in the regulation of origin activation.
Department of Cellular and Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492.
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EP
Carmichael
J M
JM
Roome
A F
AF
Wahl
D001483:Base Sequence; D002453:Cell Cycle; D016384:Consensus Sequence; D004249:DNA Damage; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D051716:Replication Protein A; D013539:Simian virus 40 / Q000235:genetics*; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A