Download Identifying Inhibitors of Y-family Polymerases Undergraduate Category: Physical and Life Sciences

Undergraduate
Category: Physical and Life Sciences
Degree Level: Undergraduate
Abstract ID#: 1004
DNA Damage Recognition
and Tolerance Laboratory
Identifying Inhibitors of Y-family Polymerases
John Lambert, Nicole Antczak, Emmanuel Zlibut, Brenna Shurtleff, Penny Beuning
Abstract
Results
DNA damage is ubiquitous, occurring on a scale of approximately 100,000 lesions per
cell per day. DNA damage can result from exposure to industrial chemicals, ultraviolet
light, ionizing radiation, thermal disruption, chemotherapy and other agents. Most
damage gets repaired; however, some damage requires specialized DNA polymerases
that can copy the damaged DNA in a process called translesion synthesis. These
polymerases belong to the Y-family and are conserved in all domains of life. We focus
on one of the two E. coli proteins, DinB and its human ortholog polymerase kappa,
which is one of several Y-family members in humans. These Y-family DNA
polymerases have poor efficiency and accuracy compared to replicative DNA
polymerases. Y-family DNA polymerases in bacteria have been implicated in antibiotic
resistance, while those in humans are implicated in cancer and decreased efficacy of
chemotherapy. A previous molecular modeling screen with a library of compounds
found 11 compounds that could be potential inhibitors. Using primer extension
assays, preliminary results have shown that most of these compounds require a
higher concentration to inhibit polymerase kappa than DinB on both undamaged and
damaged DNA. We are currently refining these results and exploring a larger range of
concentrations in order to find the IC50 of these compounds for both proteins. We
plan to test the accuracy of the polymerases in the presence of these compounds,
and test the stability of the proteins in the presence of both DNA and the compounds
to measure their binding to the polymerases.
4 of the 11 compounds remain for initial testing. Preliminary results
indicate most of the compounds tested so far are more inhibitory
to DinB than kappa.
Introduction
Damage to DNA results in covalent modification of bases, and
occurs constantly. Environmental damage comes from a wide
variety of agents, which includes chemicals, radiation, and
chemotherapy. Translesion synthesis is a mechanism for
polymerization of DNA past a lesion. Unlike other systems that act
on damaged DNA, translesion synthesis is potentially mutagenic.
The polymerases used in this work belong to the Y-family. We
focus on Escherichia coli DinB and human DNA polymerase kappa.
• Y-family DNA polymerases in bacteria contribute to antibiotic
resistance
• Humans Y-family polymerases decrease the efficacy of
chemotherapy.
Pol
kappa
DinB
PDB:
2W7O
PDB:
4IR9
Method
• Samples are made at with either undamaged DNA or lesioned
DNA templates, fluorescent primer DNA, and the polymerase
being studied (DinB or polymerase kappa).
• The timepoints are added to an electrophoreses gel for 4 hours,
then the fluorescence is measured and level of inhibition is
analyzed.
• At present time, 7 of the 11 compounds identified as potential
inhibitors have gone through initial trials.
DNA and
pol
dNTPs
Timepoints w/ quench
buffer
0
20
60
Analyze by gel
electrophoresis
Conclusions and future work
Fam
Fam
Compounds: Glycolic Acid, Pamoic Acid, Phenyl Phosphate, 2hydroxy-5-nitrobenzoic acid, aurintricarboxylic acid, D-Galacturonic
acid, glycinamide, 3-nitrosalicylic acid, ellagic acid, MK-886
• 4 of the 11 compounds remain for initial testing. Preliminary
results indicate most of the compounds tested so far are more
inhibitory to DinB than kappa.
• The accuracy of the polymerases in the presence of these
compounds will be tested, as well as their stability. This will
enable us to measure their binding to the polymerases.
References:
1. Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Shultz, R. A., and Ellenberger, T. (2006)
DNA Repair and Mutagenesis, 2nd Ed, ASM press, Washington DC
2. C. Guo, et al., Cell Mol Life Sci 2009, 66, 2363-2381.