Effect of the DinG helicase on genome instability of G quartet sequences: Determination and

Analysis of Mutation Rates

Glen Hamman

Jan Varada

Research Goals

Biological:– Determine the effect of the DinG helicase on the

genetic stability of a quadruplex-forming DNA sequence from the human RET oncogene.

Mathematical:– Develop a rigorous model for the calculation of

mutation rates.

– Luria and Delbrück, in 1943, presented the mutation rate problem. It remains unsolved.

What Are Mutation Rates?• Mutation - A permanent change in an organism’s

genotype.

• Number of mutations, not mutants.

• Calibrated per generation (time in some way)

• Mutations/cell/generation

• Provides more insight into the biology of the organism because you learn about what is happening each time the cell divides

Problem with Mutation Rates

• Experimental design doesn’t allow to keep track of cell generations accurately.

• Hard to estimate rates using only the final distribution of mutants.

• Different methods available to compute the estimates. No way to tell which one is more accurate.

• Different methods give different results.

Luria and Delbrück Experiment

Luria, S.E.,Delbrück, M. 1943 Genetics 28: 491

Mutations give rise to different numbers of mutants dependent on when the

mutation occurs

Two mutations give 4 mutants One mutation gives 16 mutants

The culture with the fewer mutants has the higher mutation rate!

Molecular Biology Primer

• DNA is made up of 4 nucleotides: A, T, C, G

• Pairing rules: A-T, C-G

• Replication: Process by which the DNA makes a copy of itself.

• Plasmids: Extra-chromosomal DNA capable of replicating independently of the bacterial chromosomal DNA

Experimental Methods

The CAT (Cm) and TET genes in plasmid pBR325 are used for genetic selection.

Quadruplex (4 stranded) DNA structures can form in G-rich DNA sequences.

Table of Mutation Frequencies

Conclusion: mutations in hfq and dinG increase mutation rates. Therefore, these helicases may be removing quadruplex structures in cells.

Strain pBR325

Cm gene

pBR235

Cm gene

BW25113 1.66E-7 4.67E-7

JW0784-1 dinG 4.29E-7 1.03E-6

Wt/mutant ratio ~ 2.58 ~ 2.20

pBR325

Tet gene

pBR235

Tet gene

BW25113 2.90E-8 9.91E-9

JW0784-1 dinG 1.26E-7 6.19E-7

Wt/mutant ratio ~ 4.38 ~ 6.25

Biological Conclusions

• Successfully cloned RET oncogene quadruplex-forming (G4) sequence into 2 selectable genes (TET and CAP) in 4 different bacterial strains. Also -- reversed the orientation of the direction of replication in these plasmids.

• DinG helicase reduce the frequency of deletions of quadruplex-forming DNA in E. coli.

This suggests that these quadruplex sequences are forming alternative DNA structures in cells

• The frequency of deletions is different for different directions of replication through the G4 sequence.This suggests that the quadruplex forms with different probability in the leading or lagging strands of replication.

Mathematical Model

• µ = E(NMn+1)/(2*E(Xn))

• = NMn+1 / (2Xn)

• NMn+1= Mn+1- 2Mn

• Xn= 2n - Mn

• = (Mn+1- 2Mn) / [2*(2n - Mn)]

• = (Mn+1- 2Mn) / (2(n+1) - 2Mn)

Mathematical Model

A manuscript is in preparation for GENETICS entitled: ON AN UNBIASED ESTIMATOR OF MUTATION RATES

R. Sinden, J.H. Dshalalow, G. Hamman, and J.C. VaradaThis paper will provide the first rigorous solution to the mutation rate problem since description

of the problem by Luria and Delbruck in 1949.€

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Future Work

• Design an experimental approach to apply our mathematical model.

• Extend the model to take into account any generation instead of consecutive ones.

• Compare the rates obtained using our model with the rates obtained using the methods from the literature.

Support and Acknowledgements

• National Science Foundation

• FIT BioMath Faculty:– Dr. Semen Koksal (PI)– Dr. Jewgeni H Dshalalow – Dr. Richard R. Sinden– Dr. David Carroll– Dr. Robert van Woesik

Reversing the direction of the AMP gene and the ColE1 replication origin in plasmid pBR325 creates plasmid pBR235. This changes the relationship between gene sequences

and the leading and lagging strands of replication.

Table of Mutation Frequencies

Strain pBR325 pBR235 235/325Cm gene Cm gene ratio

BW25113 1.66E-7 4.67E-7 ~ 2.8JW0784-1dinG 4.29E-7 1.03E-6 ~ 2.4

pBR325 pBR235 235/325Tet gene Tet gene ratio

BW25113 2.90E-8 9.91E-8 ~ 3.4JW0784-1dinG 1.26E-7 6.19E-7 ~ 4.9

Conclusion: mutation rates are different in pBR325 and pBR235. Therefore, quadruplex structures may be forming differentially in the leading and lagging strands during plasmid replication.