Mutations play a dual role in life, bad and good. An understanding of the biochemical basis of mutation is fundamental to human health issues involving genetic disorders including birth defects, heritable and sporadic cancers, neurodegenerative disorders and numerous age-related diseases. Mutations, however, also play an essential role during evolution by ensuring competitive fitness at cellular and whole organism levels, including the generation of antibody diversity in higher vertebrates. An understanding of DNA polymerase fidelity is at the core of understanding how mutations are generated. We have been studying the biochemical and physical chemical basis of DNA polymerase fidelity for the past thirty-nine years. By studying high and low fidelity DNA polymerases, we have developed concepts and methods to analyze base selection, exonuclease proofreading, and translesion DNA synthesis (TLS). While investigating the biochemical basis of SOS damaged-induced mutagenesis in E. coli, we discovered DNA polymerase V, a founding member of a new family (Y-family) of """"""""error-prone"""""""" DNA polymerases. We showed that Pol V is a heterotrimer (UmuD22C) of two proteins required for UV mutagenesis. In 2009, we resolved a long-standing issue in DNA damage-induced mutagenesis in E. coli, the direct role of a RecA nucleoprotein filament (RecA*) in the replication of damaged DNA templates by Pol V. We showed that the role of RecA* is to transfer a molecule of RecA-ATP from its 32-end to convert inactive Pol V into mutagenically active Pol V Mut (UmuD22C-RecA-ATP). The properties of Pol V Mut are regulated through a biochemical cycle of polymerase activation, TLS, deactivation and reactivation. Using bulk solution studies, we showed that RecA-ATP remains bound to UmuD22C in both activated and deactivated forms of Pol V Mut. Based on these studies, we proposed that Pol V Mut behaves as a conformational switch, with RecA-ATP changing positions relative to UmuC and UmuD22 in activated and deactivated states. Our competing renewal grant contains two major aims.
Both aims use state-of-the-art laser single molecule microscopy to visualize fundamental polymerase behavior, in vitro and in living cells, as the polymerase performs its functions in real-time.
In Aim 1, we use Total Internal Reflectance Microscopy (TIRF) in conjunction with Forster Resonance Energy Transfer (FRET) to watch, in real- time, as a single molecule of Pol V Mut makes transitions between activated and deactivated states, while synthesizing DNA, or while remaining quiescent in the absence of template DNA.
In Aim 2, we use live cell imaging to watch, in real-time, as a single molecule of Pol V, Pol II or Pol IV exchanges with the replicative DNA polymerase III at a replication fork that has stalled in the presence of DNA damage.
Each aim i n our proposal addresses an important new model.
Aim 1 addresses a new model for the regulation of DNA damaged-induced mutagenesis, where active and inactive forms of DNA polymerase are determined by an unprecedented on-off toggle switch mechanism, reflecting conformational changes of RecA-ATP within Pol V Mut. This new regulatory mechanism can act to ensure that error-prone Pol V Mut cannot mutate the cell unnecessarily by copying undamaged DNA templates.
Aim 2 addresses a new model for Pol V-Pol III exchange at a blocked replication fork, where we have found that the ? proofreading subunit of Pol III undergoes rapid degradation dependent on the induction of Pol V in living cells. This new polymerase exchange model suggests the possibility that Pol V gains access to damaged DNA by """"""""attacking"""""""" a stalled Pol III at a blocked replication fork.
In all organisms including bacteria and humans, mutations are generally harmful, causing numerous sporadic and inherited diseases. On the other hand, mutations are required for evolution and play an essential role ensuring immunological diversity and general cell and organismal fitness. The proposed research explores the biochemical mechanisms of a new type of error-prone DNA polymerase, which is activated when needed to copy damaged DNA, deactivated to keep it from mutating undamaged DNA, then reactivated again to deal with further DNA damage. This study explores biochemical mechanisms that govern the ability of error-prone DNA polymerases to copy damaged DNA that would otherwise cause a cessation of chromosome replication resulting in cell death.
|Goodman, Myron F (2016) Better living with hyper-mutation. Environ Mol Mutagen 57:421-34|
|Goodman, Myron F; McDonald, John P; Jaszczur, Malgorzata M et al. (2016) Insights into the complex levels of regulation imposed on Escherichia coli DNA polymerase V. DNA Repair (Amst) 44:42-50|
|Jaszczur, Malgorzata; Bertram, Jeffrey G; Robinson, Andrew et al. (2016) Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword. Biochemistry 55:2309-18|
|Oertell, Keriann; Harcourt, Emily M; Mohsen, Michael G et al. (2016) Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity. Proc Natl Acad Sci U S A 113:E2277-85|
|Gruber, Angela J; Erdem, Aysen L; Sabat, Grzegorz et al. (2015) A RecA protein surface required for activation of DNA polymerase V. PLoS Genet 11:e1005066|
|Robinson, Andrew; McDonald, John P; Caldas, Victor E A et al. (2015) Regulation of Mutagenic DNA Polymerase V Activation in Space and Time. PLoS Genet 11:e1005482|
|Donigan, Katherine A; McLenigan, Mary P; Yang, Wei et al. (2014) The steric gate of DNA polymerase Î¹ regulates ribonucleotide incorporation and deoxyribonucleotide fidelity. J Biol Chem 289:9136-45|
|Goodman, Myron F (2014) The discovery of error-prone DNA polymerase V and its unique regulation by RecA and ATP. J Biol Chem 289:26772-82|
|Erdem, Aysen L; Jaszczur, Malgorzata; Bertram, Jeffrey G et al. (2014) DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase. Elife 3:e02384|
|Vaisman, Alexandra; McDonald, John P; Noll, Stephan et al. (2014) Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli. Mutat Res 761:21-33|
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