Mechanism of DNA binding and localized strand separation by Pur alpha and comparison with Pur family member, Pur beta.
Pur alpha is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Pur alpha unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Pur alpha are essential for both ss- and duplex DNA binding. Pur alpha binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Pur alpha since removal of Pur alpha in the gel eliminates the series and since Pur alpha binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Pur alpha binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Pur beta lacking the Pur alpha C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Pur alpha can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Pur alpha.
Wortman
Margaret J
MJ
Department of Pathology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Johnson
Edward M
EM
Bergemann
Andrew D
AD
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017895:Manganese Compounds / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D010087:Oxides / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011687:Purines / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D016615:Telomere / Q000648:ultrastructure
D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D017895:Manganese Compounds; D009419:Nerve Tissue Proteins; D009841:Oligonucleotides; D010087:Oxides; D016271:Proto-Oncogene Proteins c-myc; C121485:Pura protein, mouse; C108770:Purb protein, mouse; D011687:Purines; D011993:Recombinant Fusion Proteins; C048856:permanganic acid; D004247:DNA; D012685:Sepharose; D001120:Arginine; D005982:Glutathione Transferase