Structural and functional studies of DNA polymerase I enzymes

We determined n 1995 the crystal structure of an N-terminal deletion of T. aquaticus (Taq) DNA polymerase I (Korolev et al., 1995). This fragment correspond to the "klenow" fragment of the polymerase. This structure reveals the structural basis for thermostability and has been the stepping stone onto which we have developed a program aimed at addressing the issues of nucleotide incorporation and recognition. In a first step taken to address those issues, the structures of binary complexes of the polymerase with dNTPs (N=A,T,C,or G) were solved. These structures, refined to a resolution of 2.3 A, identified the O helix as the binding site for dNTPs and show that the dNTPs can bind in a conformation favorable to incorporation (Li et al.,1998a ). Next, a ternary complex of the polymerase with a primer/template DNA reacted to ddCTP was examined. In the process of determining this structure, a binary complex of the polymerase with the same primer/template DNA (without ddCTP) was obtained and solved. The two structures differ by a large conformational change affecting the tip of the fingers domain with the O helix rotating inwards (close conformation) towards the active site. This conformational change is believed to be essential to deliver the dNTPs to the active site. These structures compared to the apo form were also useful in characterizing the conformational changes required upon binding of a primer/template DNA (Li et al.,1998b ; also see movie by clicking on the animated picture above). More recently, we have solved the structures of three additional ternary complexes corresponding to the ddATP, ddGTP, and ddTTP-trapped forms of the polymerase. We observed that the ddGTP-trapped form differs from the other three forms in that a side chain in the O helix makes additional H-bond interactions with the G base of ddGTP. We hypothesized that these interactions are responsible for the higher rate of incorporation of ddGTP compared to the three other ddNTPs which Brandis et al. (1996) observed for the Taq polymerase. To test this hypothesis, we mutated this residue and discovered that mutations at this position selectively affect the rate of incorporation of ddGTP without affecting ddATP, ddCTP, or ddTTP incorporation rates (see details in Li et al.,1999 ). The structures of the four ddNTP-trapped Klentaq1 complexes were also analyzed in an in-depth comparison study that sheds light into the mechanism of nucleotide incorporation and selectivity (see details in Li et al.,2001 ).

One fundamental debate in the field of DNA polymerase enzyme has been whether the large conformational transition affecting the fingers subdomain upon addition of the correct dNTP that we and others have characterized structurally (see above) corresponds to the rate-limiting step established by kinetic studies and known to be essential in nucleotide discrimination. We have sought to settle this issue by setting up a FRET (fluorescence resonance energy transfer) system that monitors conformational changes in the fingers subdomain. We successfully coupled acceptor and donor fluorescence probes to the protein and the DNA, respectively, and shown that a FRET signal corresponding to the closing of the fingers domain can be reliably observed. This allowed us to measure the rate of dNTP-induced closing of the fingers subdomain and compare it to the rate of the rate-limiting step of nucleotide incorporation (termed kpol) (for details, see Rothwell et al. (2005)). We found that fingers subdomain closure is fast compared to kpol, demonstrating that fingers subdomain closure is not rate-limiting. Using the same FRET system, we uncovered a pre-equilibrium preceding closure (see details in Rothwell et al. (2007)). Recently, we set up an intramolecular FRET system that allowed monitoring of fingers subdomain opening (details in Allen et al. (2008)) and showed that fingers opening is limited by kpol.

Explore the following links to learn about other projects related to DNA replication: E.coli SSB, The Rep helicase, and Phage P4 OBD .

Publications