Pol-4 catalyzes replication of the genome and other DNA synthetic processes in human cells. However, the precise functions of Pol-4 in replication, in repair of endogenous and environmental damage, and in recombination remain to be defined. Further, we lack information on the role of somatic mutations in Pol-4 in tumorigenesis and other disease processes. Our goals are to identify the functions of Pol-4 in DNA synthesis and the possible role of increased mutation by Pol-4 in the generation of human cancer. A major approach will be to exploit mutants of Pol-4 that incorporate mutagenic nucleotide analogs and thereby serve as tools to identify DNA synthesized by Pol-4 in vivo. We have four specific aims.
In Aim 1, we will generate mutants of Pol-4 that increase incorporation of a specific nucleotide analog(s).
In Aim 2, we will purify wild-type and mutant Pol-4 holoenzyme complexes and characterize their catalytic properties in detail, including their fidelity and kinetics of analog incorporation.
In Aim 3, we will define the roles of Pol-4 in mammalian cells by introducing mutant Pol-4's that preferentially incorporate mutagenic nucleotide analogs and measuring induced mutation in cells undergoing DNA replication, repair and recombination. The induced mutations will identify the DNA synthesized by Pol-4.
In Aim 4, we will assess the role of increased mutagenesis in tumor progression by performing serial transfer experiments to determine if mammalian cells that harbor mutator Pol-4 have a competitive advantage, and if there are nucleoside analogs that diminish this advantage.
Our objective is to establish the roles of DNA polymerase-4 in replication of the human genome and in repair of damage caused by endogenous and environmental agents. We will determine if mutations in DNA polymerase-4 promote genetic instability and accelerate tumor progression in model systems.
We aim to identify a new class of chemotherapeutic agents that will retard tumor growth.
| Beckman, Robert A; Loeb, Lawrence A (2017) Evolutionary dynamics and significance of multiple subclonal mutations in cancer. DNA Repair (Amst) 56:7-15 |
| Fox, Edward J; Reid-Bayliss, Kate S; Emond, Mary J et al. (2014) Accuracy of Next Generation Sequencing Platforms. Next Gener Seq Appl 1: |
| Fox, Edward J; Loeb, Lawrence A (2014) Cancer: One cell at a time. Nature 512:143-4 |
| Kennedy, Scott R; Schmitt, Michael W; Fox, Edward J et al. (2014) Detecting ultralow-frequency mutations by Duplex Sequencing. Nat Protoc 9:2586-606 |
| Shen, Jiang-Cheng; Fox, Edward J; Ahn, Eun Hyun et al. (2014) A rapid assay for measuring nucleotide excision repair by oligonucleotide retrieval. Sci Rep 4:4894 |
| Weedon, Michael N; Ellard, Sian; Prindle, Marc J et al. (2013) An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy. Nat Genet 45:947-50 |
| Kennedy, Scott R; Salk, Jesse J; Schmitt, Michael W et al. (2013) Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage. PLoS Genet 9:e1003794 |
| Fox, Edward J; Prindle, Marc J; Loeb, Lawrence A (2013) Do mutator mutations fuel tumorigenesis? Cancer Metastasis Rev 32:353-61 |
| Prindle, Marc J; Schmitt, Michael W; Parmeggiani, Fabio et al. (2013) A substitution in the fingers domain of DNA polymerase ? reduces fidelity by altering nucleotide discrimination in the catalytic site. J Biol Chem 288:5572-80 |
| Schmitt, Michael W; Kennedy, Scott R; Salk, Jesse J et al. (2012) Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci U S A 109:14508-13 |
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