Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase

Abstract

T4 phage polynucleotide kinase (PNK) displays 5'-hydroxyl kinase, 3'-phosphatase and 2',3'-cyclic phosphodiesterase activities. The enzyme phosphorylates 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 oligonucleotide substrates of various lengths and sequences. In these structures, the 5' ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first 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 substrate molecule.

Figure 1

Composite omit maps of the DNA binding region and kinase active site. (a) Observed density for the 5′-GTCAC-3′ substrate. The second base (thymine) is stacked against the 5′ guanine and forms a polar aromatic interaction with Y52. The edges of both bases are solvent-exposed, and the opposite face of the guanine base is flanked by non-polar residues in the base of the active site. (b and c) The density for the 5′-TGCAC-3′ substrate [(b) is in the same orientation as (a); (c) is rotated 90° relative to (b) to better visualize the stacking interaction between the second and third bases]. The 5′ thymine base and its ribose sugar are located in a similar position as the 5′ guanine on the previous panel. The second base (guanine) has swung out of the active site cleft and is stacked against the third base (cytosine). The structural roles of several side chains in DNA binding are different between the two complexes.

Figure 2

Schematic of interactions to the first three ordered bases in the PNK–DNA crystal structures. The most conserved protein–DNA contacts between the two sequences of DNA substrates are to Val 131 (which contacts the 5′ base in both structures), D35 and T86 (which contact the 5′ ribose sugar and its adjoining phosphate non-binding oxygen). Both residues are known to be essential for catalysis in PNK mutagenesis studies, with the exception of a T86S substitution. One methionine residue, which is actually a selenomethionine in these structures, is observed to make a long contact between its terminal methyl group and the thymine nucleotide in the TGCAC complex. However, the specific activity of the selenomethionyl enzyme is not reduced relative to the wild-type enzyme (data not shown).

Figure 3

Conformation of bound 5′-GTC-3′ and ADP (A) or 5′-TGC-3′ and ADP (B) to the PNK domain. The nucleotide phosphate donor is bound on the opposite side of the tunnel formed by the interactions of two loops that extend over the 5′ nucleotide sugar. Protein orientation is the same in both panels.

Figure 4

Superposition of bound 5′-GTC-3′(blue) and 5′-TGC-3′ (gray) and surrounding protein residues (green and magenta, respectively). Note the flip of the second base in the 5′-TGC-3′ structure, and its position relative to the third (cytosine) base in the 5′-GTC-3′ structure.