During DNA replication, DNA polymerases follow an induced fit mechanism in order to rapidly distinguish between correct and incorrect dNTP substrates. The dynamics of this process are crucial to the overall effectiveness of catalysis. Although X-ray crystal structures of DNA polymerase I with substrate dNTPs have revealed key structural states along the catalytic pathway, solution fluorescence studies indicate that those key states are populated in the absence of substrate. Herein, we report the first atomistic simulations showing the conformational changes between the closed, open, and ajar conformations of DNA polymerase I in the binary (enzyme∶DNA) state to better understand its dynamics. We have applied long time-scale, unbiased molecular dynamics to investigate the opening process of the fingers domain in the absence of substrate for B. stearothermophilis DNA polymerase in silico. These simulations are biologically and/or physiologically relevant as they shed light on the transitions between states in this important enzyme. All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies. Furthermore, we have detailed the key stages in the opening process starting from the open and ajar crystal structures, including the observation of a previously unknown key intermediate structure. Four backbone dihedrals were identified as important during the opening process, and their movements provide insight into the recognition of dNTP substrate molecules by the polymerase binary state. In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics. All organisms are dependent on the proper replication of their DNA for survival. DNA polymerase is the enzyme responsible for copying our DNA during cell division. We have performed computational simulations on DNA polymerase to understand the fundamental dynamics of the enzyme. Our simulations provide new information about the way polymerase moves in solution that is not obtainable through traditional experimental techniques. In particular, we investigated the dynamics of DNA polymerase “opening” in the binary state (enzyme+DNA with no nucleotide substrate) starting from three different conformations. The results are consistent with available experimental data on the relative conformations of DNA polymerase in the binary state. Furthermore, we identified a novel intermediate species that we hypothesize plays a role in the dynamics of nucleotide substrate binding. Additionally, we determined the previously unknown ordering of events in the opening mechanism, and suggest new details about how the polymerase may interact with an incoming nucleotide substrate. Lastly, this research serves as a proof of principle that we can use our methodology to perform long-time scale computational simulations on DNA polymerase to explain currently unknown phenomena surrounding DNA replication.

Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary (E–DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5′-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.

Rifampicin is an effective antibiotic against mycobacterial and other bacterial infections, but resistance readily emerges in laboratory and clinical settings. We screened Escherichia coli for rifampicin resistance and identified numerous mutations to the gene encoding the β-chain of RNA polymerase (rpoB), including an unusual 9-nucleotide deletion mutation. Structural modeling of the deletion mutant indicates locations of potential steric clashes with rifampicin. Sequence conservation in the region near the deletion mutation suggests a similar mutation may also confer resistance during the treatment of tuberculosis.

Certain species D human adenoviruses (HAdV-D19, -D37, and -D64) are causative agents of epidemic keratoconjunctivitis. HAdV-D37 has previously been shown to bind CD46 (membrane cofactor protein) and sialic acid as adhesion receptors. HAdV-D64 is genetically highly similar to HAdV-D37, with an identical fiber protein sequence, but differs substantially in its penton base and hexon proteins, two other major capsid components, due to genetic recombination. Here, we demonstrate that, like HAdV-D37, HAdV-D64 virions bind directly to CD46 and that CD46 and sialic acid also function as receptors for HAdV-D64 on multiple cell types. Expression of CD46 on CD46-negative cells conferred susceptibility to HAdV-D64 entry. Specifically blocking HAdV-D64 binding to CD46 on the host cell surface strongly inhibits viral entry and gene delivery into multiple cell lines that represent target tissues. We show that CD46 is expressed on human conjunctival epithelial cells and directly binds to the HAdV-D64 virion. Our results suggest that HAdV-D64 may be used to deliver genes to target conjunctival cells and that interrupting HAdV-D64 entry through its interaction with CD46 may prevent or lessen adenovirus-associated ocular disease.