Helix-destabilizing properties of the human single-stranded DNA- and RNA-binding protein Puralpha.
Puralpha is a ubiquitous nucleic acid-binding protein which has been implicated in the control of eukaryotic gene transcription. Further, Puralpha associates with DNA sequences positioned in close proximity to viral and cellular origins of replication suggesting a role for this protein in DNA replication. As initiation of transcription and replication require alteration in the structure of duplex DNA, we investigated the DNA unwinding activity of this single-stranded nucleic acid-binding protein. Here we demonstrate that Puralpha has the ability to displace an oligonucleotide annealed to single-stranded M13 DNA. The helix unwinding activity of Puralpha was dose-, time- and temperature-dependent and ATP-independent. Results from mapping studies revealed that the central region of Puralpha, spanning amino acids 72-274, was important for the helix-destabilizing activity of this protein. The region of Puralpha which was involved in the helix-destabilizing activity mapped to the DNA-binding domain of this protein. Results from heat inactivation experiments demonstrated that the helix-destabilizing activity of Puralpha correlates with its capacity to interact with DNA containing the PUR element. Taken together, these studies demonstrate that Puralpha possesses helix-destabilizing activity and that this activity maps to and correlates with its ability to interact with DNA.
Darbinian
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Laboratory of Molecular NeuroVirology, Center for NeuroVirology and Cancer Biology, College of Science and Technology, Temple University, 1900 N. 12th Street, 015-96, Philadelphia, PA 19122, USA.
Gallia
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Khalili
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D000255:Adenosine Triphosphate / Q000378:metabolism; D017104:Bacteriophage M13 / Q000737:chemistry / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; C501130:PURA protein, human; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D005982:Glutathione Transferase