Speaker: Zucai Suo, Ph.D.
        Principal Investigator, Department of Chemistry and Biochemistry， The Ohio State University
        Web:http://chemistry.osu.edu/~suo.3/index.html
Time: 15:00 pm, 20th June (Tuesday)
Venue: Room 300, SIBS Main Building, Yueyang Road 320
Host: Dr. Zefeng Wang
        CAS-MPG Partner Institute for Computational Biology

Title: How is DNA synthesized by DNA polymerases?

Abstract:
Although lamivudine and emtricitabine, two L-deoxycytidine analogs, have been widely used as antiviral drugs for years, it is structrually unknown how they are bound and incoproated by any DNA polymerases or reverse transcriptases. To fill the void, we solved 12 high resolution ternary crystal structures of human DNA polymerase lambda, DNA, and L-dCTP or the triphosphates of lamivudine and emtricitabine. The structures of these 12 ternary complexes reveal that relative to natural D-dCTP in the canonical ternary structure, these L-nucleotides all have their ribose rotated by 180°. Among the four ternary complexes with a specific L-nucleotide in each asymmetric unit, two are similar and show that the L-nucleotide forms three Watson–Crick hydrogen bonds while in the remaining two similar ternary complexes, the L-nucleotide surprisingly interacts with the side chain of a conserved active site residue R517 through one or two hydrogen bonds. Our mutagenic and kinetic studies further demonstrate that the side chain of R517 is critical for L-nucleotide binding and incorporation.

One common oxidative DNA lesion, 8-oxoG, is highly mutagenic in vivo due to its anti-conformation forming a Watson-Crick base pair with correct dCTP and its syn-conformation forming a Hoogsteen base pair with incorrect dATP. Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson-Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg(II) appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. Our "three-metal-mechanism" overthrows the dogma of "two-metal-mechanism" in the field of polymerases. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3'-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures of how a polymerase correctly and incorrectly bypasses a DNA lesion.

All are welcome!