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Dear Ed,
I am starting work on a protein that binds double-stranded DNA. In other dsDNA-protein stuctures I have noticed that some structures use blunt-ended DNA while others use sticky end DNA with 1,2 or 3 base G/C overhangs. Is there an advantage to using sticky-end vs. blunt-end DNA for crystallization? Also, what is the reason for using the G/C overhang?
One common crystallization contact in a significant fraction of protein/DNA cocrystals is end-to-end packing of DNA ends. This idea can be used to design in DNA crystal contacts using complementary single base overhangs such G/C to allow for Watson-Crick base pairing.
Where this gets really interesting is when you also take into consideration (a) triple-strand formation instead of simple Watson-Crick base pairing and (b) potential DNA bending intrinsic to the DNA fragment or induced by the DNA-binding protein. We published a paper which explores these ideas a few years ago:
Tan, S., Hunziker, Y., Pellegrini, L. and Richmond, T.J. (2000) Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization, J. Mol Biol., 297: 947-959.
A reprint of this paper is available at http://www.bmb.psu.edu/faculty/tan/lab/reprints.html .
An important point to keep in mind is that although such designed DNA crystal contacts may increase the likelihood of producing cocrystals of protein/DNA complexes, you should probaby explore different sized DNA fragments with blunt or other ends because there's no guarantee designed ends will produce the best ordered crystals. For example, critical crystal contacts have resulted from blunt DNA ends packing against the protein in many protein/DNA complex cocrystals, and such crystal contacts were not engineered.
Regards, Song -- Dr. Song Tan Associate Professor in Biochemistry & Molecular Biology Center for Gene Regulation Dept of Biochemistry & Molecular Biology 108 Althouse Laboratory (office & lab in 3 Althouse Laboratory) Penn State University University Park, PA 16802 email: [EMAIL PROTECTED] http://www.bmb.psu.edu/faculty/tan/lab/ phone: 814-865-3355 fax: 814-863-7024
