Overproduction, purification, and characterization of DNA-binding protein P19 of bacteriophage PRD1

Abstract

The early protein, P 19, of bacteriophage PRD1 was purified after overexpression of the cloned gene, XIX, in Escherichia coli DH5α cells. The purified protein binds as multimers to single-stranded DNA (ssDNA), and with a lower affinity to double-stranded DNA (dsDNA), without sequence-specificity. Two distinct P19-ssDNA complexes were discovered in gel- mobility-shift assays at different protein:DNA ratios. P 19 was capable of fully protecting ssDNA against nuclease P1. Electron microscopy of protein P19-ssDNA complexes showed DNA molecules which were extensively coated with protein and whose contour length was clearly reduced by P 19 binding. The results suggest that P 19 binds to ssDNA with moderate cooperativity and are consistent with the DNA being wrapped around the P 19 multimers.