Effect of the DinG helicase on genome instability of G quartet sequences: Determination and Analysis of Mutation Rates Jan C. Varada*1,2, Glen E. Hamman*1, Richard R. Sinden2, Jewgeni H. Dshalalow1 1 Department of Mathematical Sciences, 2Department of Biological Sciences, Florida Institute of Technology, 150 W. University Blvd. Melbourne, Florida 32901 (Contact email – [email protected]) Mutations are permanent changes in an organism’s DNA. Observable changes that occur in the DNA can be measured and a mutation rate at which these changes occur can be established by performing experiments such as the fluctuation assay performed by Luria and Delbrück (1). The question of how to generate mathematically the distributions obtained by these fluctuation assays has been addressed several times during the past fifty years with varying degrees of success (2). In this project we have taken a guanine rich sequence from the promoter region of the RET oncogene and cloned it into the plasmid vector pBR325. This oncogene has been associated with several types of cancer, and the specific sequence is known to form G-quadruplex structures that may affect the regulation and or genetic instability of this gene (3, 4). We have transformed this plasmid into the strains BW25113 and JW0784-1 of the bacteria Escherichia coli to study the possible effects of the DinG protein in the unwinding of such quadruplex structures. The DinG protein is a helicase that has been shown to unwind DNA-RNA duplexes (5, 6). Preliminary data suggests at least a 2 to 6 fold difference in the mutation rates between the strains, indicating a possible role of the DinG protein in unwinding the quadruplex structures. Furthermore it shows the possibility that G-quadruplexes are being formed in vivo in the E. coli cells. Future studies will address whether the orientation of the sequence with respect to the origin of replication is a significant factor affecting the mutation rates, and whether the methods for determining the mutation rates from the obtained distributions could be improved. 1. Luria, S. E. and Delbrück, M. (1943) Genetics 28, 491-511. 2 Rosche, W. A. and Foster, P. L. (2000) Methods 20, 4-17. 3 Guo, K., et al. (2007) J Am Chem Soc 129, 10220-10228. 4 Cogoi, S. and Xodo, L. E., (2006) Nucleic Acid Res. 34(9), 2536-2549. 5 Voloshin, O. N. and Camerini-Otero, R. D., (2007) J Biol Chem 282(85), 1834718447. 6 Voloshin, O. N., et al., (2003) J Biol Chem 278(30), 28284-28293.