Mutations are permanent changes in an organism DNA

advertisement
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.
Download