Within the last 15-20 years it has become clear that many DNA sequences in the human genome are inherently unstable in that they undergo DNA directed mutations that cause disease and cancer. Certain DNA sequences, generally repeated elements, can form alternative DNA conformations that participate in events that lead to DNA strand breaks, rearrangements, deletions, or duplications. One of these sequences that can form a four stranded DNA structure is termed quadruplex DNA (see Sinden, 1994, Chapter 7 (Sinden, 1994)). DNA sequences that form quadruplex structures have been identified as a sequence element of many human genes (Eddy and Maizels, 2008; Maizels, 2006; Eddy and Maizels, 2006). Moreover, many oncogenes, genes that when mutated lead to progression and formation of cancer cells, contain quadruplex-forming DNA sequences (Duquette et al., 2007; Qin and Hurley, 2008).
The Sinden laboratory has developed a mutational selection system that allows measurement of rates of DNA-directed mutation. This involves the insertion of DNA repeats into the chloramphenicol acetyltransferase (CAT) gene. DNA insertions usually render the gene inactive resulting in a chloramphenicol sensitive (Cms) phenotype. Reversion to chloramphenicol resistance (Cmr) occurs by loss (deletion) of all or part of the inserted DNA repeats. Differences in deletion rates can occur from orientation differences of the repeats because alternative DNA secondary structures can form and these form at different rates in the leading or lagging strands of replication. We have used this system to study the stability of inverted repeats, quasipalindromes, direct repeats, and repeats associated with neurodegenerative diseases (Rosche et al., 1997; Rosche et al., 1995; Trinh and Sinden, 1991; Hashem and Sinden, 2005; Hashem et al., 2004; Hashem et al., 2002; Sinden et al., 1991).
The genetic stability of quadruplex DNA structures has not been analyzed in a model mutational analysis system. The project the BIOMATH students have undertaken is an ambitious one that will address the important question of the genetic instability of DNA sequences associated with the oncogenes that can form G-quartets (quadruplex DNA). Previous Biomath students, Jan Varada and Glen Hamman, cloned chemically synthesized G-quartet-forming sequence from the RET oncogene into both the CAT gene as well as the tetracycline resistance gene in plasmid pBR325. This involves two different plasmid constructs - pBR325BG4 and pBR325EG4. They then have reversed the origin of replication (making derivatives they have named pBR235) for these two plasmids (making 2 additional plasmids - pBR235BG4 and pBR235EG4). These plasmids were transformed into 4 bacterial strains: MC4100 (wild-type) and Hfq- (an isogenic pair with a mutation in the hfq gene, and BW25113 (wild-type) and JW0784-1, a containing dinG mutation. Both the DinG and Hfq proteins are suspected of being proteins that may act to remove quadruplex DNA structures from DNA. Mutation rates were then measured for 10 strains (BW25113 containing pBR325BG4, pBR325EG4, pBR235BG4, and pBR235EG4; JW0784-1 containing pBR325BG4, pBR325EG4, pBR235BG4, and pBR235EG4; and MC4100 and Hfq- containing pBR325BG4 and pBR235BG4). The results agree with the hypothesis being tested that mutations in genes that remove quadruplex DNA structure from DNA will increase mutation rates. This result is highly significant, in that it demonstrates for the first time that quadruplex structures are forming inside cells.
Current BIOMATH students Brittany Niccum and Marisa Rivera have continued this line of work. Their approach has been to clone quadruplex-forming DNA sequences that form quadruplexes more readily than the RET oncogene sequence (Risitano and Fox, 2003; Kumar et al., 2008). These sequences are shown in Figure 1.
Cloning into the chloramphenicol acetyltransferase gene in plasmid pBR325 allows the measurement of the rate of deletion of the quadruplex sequence from the DNA. They have also reversed the orientation of the unidirectional origin of replication in these plasmids, creating pBR235 plasmids. Thus, in plasmids pBR325 and pBR235 the G-tracts are on either the leading or lagging strand of DNA replication. These plasmids have been transformed into Escherichia coli strains BW25113 and JW0784-1 to study the possible effects of the DinG protein in the unwinding of such quadruplex structures. The DinG protein is a helicase that can reportedly unwind unusual DNA structures. Some of the quadruplex-forming sequences are genetically very unstable in cells, and initial data show a 40-fold ratio comparing sequences in pBR325 and pBR235. The results suggest that quadruplexes are forming in vivo in the E. coli cells in a leading or lagging strand specific fashion. Future analysis will determine if the DinG helicase is involved in removing quadruplexes.
The final step in this analysis is to undertake a through mathematical analysis of mutation rates using the methods reviewed by Rosche and Foster (Rosche and Foster, 2000). The analysis of mutation rates is a complex mathematical analysis given a random, stochastic process. The number of mutants in a population will vary depending on when the mutation occurs during growth of the population. This problem has given experimentalists great difficulty since the problem was described by Luria and Delbruck in 1943 (Luria and Delbruck, 1943) scientific field (Rosche and Foster, 2000; Foster, 2006). With Dr. Dshalalow and Sinden, the previous Biomath students have developed a stochastic model for mutation rate analysis: Sinden, R.R., Dshalalow, J.H., Hamman, G.E., & Varada, J.C. On an Unbiased and Consistent Estimator of Mutation Rates, Submitted for publication (and currently under revision):
The following formula for the unbiased and consistent estimator În of the unknown mutation rate µ holds:
Here Mn stands for the total number of mutants at the nth generation. A second publication will describe the mutation rate analysis for the hfq and the dinG mutations for many of the quadruplex sequences.
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2 - Rosche, W. A. and Foster, P. L. (2000). Determining Mutation Rates in Bacterial Populations. Methods 20, 4 - 17.
3 - Guo, K., et al. (2007). Formation of Pseudosymmetrical G-Quadruplex and i-Motif Structures in the Proximal Promoter Region of the RET Oncogene. J Am Chem Soc 129, 10220 - 10228.
4 - Cogoi, S. and Xodo, L. E. (2006). G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription. Nucleic Acid Res 34(9), 2536-2549.
5 - Voloshin, O. N. and Camerini- Otero, R. D. (2007) The DingG Protein from Escherichia coli Is a Structure-specific Helicase. J Biol Chem 232(85), 18347 - 18447.
6 - Voloshin, O. N., et al. (2003). Characterization of the DNA Damage-inducible Helicase DinG from Escherichia coli. J Biol Chem 278(30), 28284 - 28293.