Kiefer et al’s research paper, “Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal”, focused on determining a highly resolved crystal structure (at 1.8Å resolution)  of the active site of a bacterial DNA polymerase bound catalytically with DNA primer templates, so as to better understand the mechanism by which nucleotides are correctly incorporated into a replicating DNA strand. To retain the fidelity of the DNA synthesis process, DNA polymerase I from a thermophilic bacteria (Bacillus stearothermophilus) was used because of its stability at high temperatures.

Bacillus fragments with DNA primer substrates were co-crystallized using the hanging drop-diffusion method. The DNA template strand had an identical duplex nine- base pair region, and a 5’ single stranded overhang for binding of the polymerase. Diffraction data of the templates, as well as electron density maps were collected, to determine the crystal structure before and after treatment with polymerase. Catalysis of the enzyme was investigated by incubating the co-crystals in separate stabilizing solutions: the first containing ddTTP, which resulted in the addition of one newly synthesized base pair; the second incubation in dATP, causing two new base pairs to be added. The use of ddTTP is of particular significance as it acts as a chain termination nucleotide. Difference electron density maps confirmed successive translocation at positions previously occupied by the initial substrate and that the enzyme recognized mismatched base pairing. Hence, the polymerase retained its catalytic and simultaneously discriminated between correct and incorrectly paired nucleotides.

Comparison of the translocated complexes showed several common structural features.

Extensive sequence-independent interactions were observed between the protein and the first four bases of the DNA minor groove recognition (MGR) region, extending from the 3’ primer terminus. Stacking interactions caused by Tyr714, which sandwiches itself between template bases via Van der Waals forces, forms a pocket of high steric complementarity around the first base pair at the active site. The pocket provided pronounced specificity for correctly paired nucleotides. As translocation continues from one base pair position to the next, a conformational transition of the DNA strand occurs from B-form, with C2’-endo sugar pucker, to underwound A-form and C3’-endo pucker, allowing the enzyme’s side chains access to the edges of the nucleotide bases.

The actual catalytic mechanism of DNA polymerase is described and subsequently illustrated in two crystallographic figures. Within the active site, Asp 830 hydrogen bonds to the 3’OH terminus temporarily shifting the DNA template to an A-form 3’endo conformation. Once Asp 830 deprotonates the 3’OH terminus, the resulting 3’oxy-anion then attacks the incoming dNTP at the α-phosphate, and in doing so switches back to the A-form C3’endo conformation. Addition of the dNTPs are facilitated through the stabilization effects of a divalent metal cation that is associated with Asp 653 and Asp 830. This occurs because the metal cation stabilizes the anionic α-, β-, and γ-phosphates of the incoming dNTP due to close proximity. Sugar specificity (of the incoming dNTP) is attained through the close positioning of Glu 658 and Phe 710 in regards to the 3’OH perhaps due to hydrogen bonding. Mismatch prevention is also discussed at the level of the active site and is illustrated through crystallographic figures. The position of Tyr 714 at the exact location predicted for the first nucleotide of the 5’ template overhang. This causes the overhang to turn 90º and bind within a “shallow pocket” within the O-helix. The fact that such a conformation inhibits further dNTP base pairing suggests that Tyr 714 aids in preventing mismatches. All these structural changes and interactions between the active site and template resulted in greater specificity for correct nucleotide incorporation, thus maintaining the high fidelity of DNA replication.

How do the two papers relate to one another?  How did the earlier paper set the stage for the second paper?

The two papers trace the progressive understanding of the mechanism of DNA replication, by specifically focusing on the properties and function of DNA polymerase.

The Lehman paper outlined the step-by-step procedure of isolating and purifying the polymerase, as well as the preparation of substrates needed in each step.

Its detailed work set the stage for more in-depth analysis of the replication process, an example being the Kiefer paper. The work of Kiefer et al, presented a high resolution view of the active site-template strand translocated complex in crystal form and thus, gave insight into the structural interactions occurring at the polymerase active site that enable correct base pairing in DNA replication.