Scholar Profile

Lorena S. Beese

Professor
Department of Biochemistry
Duke University
Box 3711
Durham, NC 27710
Voice: 919-681-5267
Fax: 919-684-8885
Email: lsb@biochem.duke.edu
Personal Homepage
1994 Searle Scholar

Research Interests

Structural Studies of DNA Replication Proteins

The primary objective of this study is to provide a structural explanation for the fidelity and mechanism of DNA replication and an understanding of how mutations arise and are repaired. By applying the techniques of X-ray crystallography, site directed mutagenesis, and biochemical analysis we are investigating DNA polymerases complexed with a variety of DNA substrates to address these fundamental questions. In the past year we have determined the structure of a thermostable DNA polymerase from a newly identified strain of Bacillus spp with an optimal growth temperature of 72C. Its amino acid sequence is 40% identical to E. coli DNA Polymerase I, and 43% identical to the thermostable DNA polymerase from Thermus aquaticus. The crystal structure was determined by the method of multiple isomorphous replacement including the anomalous scattering data from two heavy atom derivatives. The R-factor of the refined structure is 19.8% between 8 Angstroms and 2 Angstroms resolution (Rfree=25%) with 0.010 Angstroms rms deviation in bond lengths and 1.5 _ rms deviation in bond angles.

The structure is folded into two domains. Like Klenow fragment and HIV reverse transcriptase, the polymerase domain of this Bacillus polymerase can be described by analogy to a right hand. The very highly conserved palm subdomain where DNA synthesis occurs is almost identical to that of the Klenow fragment. This structural similarity will facilitate the analysis of the structural basis for biological function as we expect to be able to apply the results from years of site directed mutagenesis studies and biochemical studies done on Klenow fragment to this polymerase. The structures differ however, in the fingers and the top of the thumb subdomains. We observe these regions to interact with DNA and speculate that they may contribute to the ten fold greater processivity of this Bacillus polymerase compared with Klenow fragment. Although the Bacillus polymerase does not have 3'-5' proofreading exonuclease activity, the overall fold of its 200 residue N-terminal domain resembles the exonuclease domain of Klenow fragment.

We have co-crystallized this polymerase with a duplex DNA primer - template and determined the structure of the complex. Both apo polymerase and DNA complex structures are determined to 2 Angstroms resolution. The polymerase-DNA co-crystals diffract to better than 1.6 Angstroms resolution on a rotating anode X-ray source which enables us to obtain the most detailed view of any polymerase-DNA complex to date.The structure of the complex shows DNA bound to the polymerase active site. This is the first co-crystal structure of a polymerase in the Pol I family with DNA is bound to the polymerase active site. It resolves a recent controversy about the direction of DNA synthesis.

The DNA enters the polymerase cleft from the direction proposed based on editing complex of the Klenow fragment. DNA adopts a primarily B-form conformation, however the minor groove widens as the DNA enters the polymerase cleft. No bend in the DNA is necessary to reach the polymerase active site. The 3' OH of the primer strand interacts with a Mg ion that is bound to a highly conserved aspartic acid residue. Mutations in this residue abolish polymerase activity. A network of hydrogen bonds is made between the sugar-phosphate backbone of the DNA and highly conserved residues of the protein. The protein undergoes a number of conformational changes upon binding DNA. Two helices forming the thumb region twist so as to widen the polymerase cleft. Helices forming the fingers region also move to widen the DNA binding cleft by several Angstroms. Residues in the catalytic triad also reposition upon binding DNA. The new structures combined with site directed mutagenesis studies suggest several residues that may have a key role in the fidelity of synthesis.

The next key experiment for understanding the fidelity of replication is to determine the structure of the polymerase with DNA substrate and nucleotide triphosphates. We have transferred polymerase:duplex DNA co-crystals into stabilizing buffer containing 10mM ddATP and 20 mM MgSO4 and determined the structure of the complex. Much to our surprise the polymerase retained catalytic activity in the crystals. The length of DNA primer increased by one nucleotide. The structures of ternary complexes including correct mismatched nucleotides are now being pursued.

Since this new thermostable Bacillus DNA polymerase is particularly amenable to structural studies and is catalytically active in our crystals we can now realistically expect to obtain structures of the different polymerase-DNA complexes essential to understanding each step of DNA synthesis.