Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase

T4 phage polynucleotide kinase (PNK) displays 5¢-hydroxyl kinase, 3¢-phosphatase and 2¢,3¢-cyclic phosphodiesterase activities. The enzyme phos-phorylates the 5 ¢ hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonu-cleotide substrates of various lengths and sequences. In these structures, the 5 ¢ ribose hydro-xyl is buried in the kinase active site in proper align-ment for phosphoryl transfer. Depending on the ssDNA length, the ®rst two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases. The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5¢-GT DNA end than for a 5¢-TG end. The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique sub-strate molecule