DNA damage blocks the progress of the replisome. Left unresolved these lesions can result in cell death. A prominent resolution mechanism is translesion synthesis (TLS), a DNA damage tolerance pathway in which an error-prone TLS polymerase switches with a high fidelity replicative polymerase to synthesize past the lesion, enabling the replisome to progress past the damage. While TLS polymerases contribute to genome stability, their misregulation has been implicated in both cancer progression and the development of antimicrobial resistance. This proposal will employ biochemical and genetic approaches in combination with in vitro and cell based single-molecule imaging to understand how TLS is regulated during bacterial replication.
Aim 1 : Elucidate the role of SSB in regulating Pol IV-mediated TLS We have demonstrated that the TLS polymerase Pol IV is recruited to replisomes in a DNA damage dependent manner through interactions with SSB. As SSB is constitutively present in the replisome this raises the question of how Pol IV is selectively targeted to stalled forks. In this aim we will work to elucidate this mechanism by determining the dynamics of SSB and the molecular events that lead to Pol IV recruitment. Finally, by using single-molecule imaging of other SSB-interacting proteins (SIPs), we will test if damage- dependent replisome recruitment is a general mechanism or if it is unique to Pol IV.
Aim 2 : Is an interaction with SSB important for recruiting TLS polymerases to their site of action? Building on our observations that the interaction of Pol IV with SSB is critical for its ability to carry out TLS, we will determine if an interaction between Pol II and SSB is also important for its function. In this aim we will develop mutants that selectively ablate the Pol II-SSB interaction. With these mutants in hand we will next ask whether the Pol II-SSB interaction is required for the recruitment of Pol II to stalled replisomes and TLS. Furthermore, we will determine the role of the Pol IV-SSB interaction in mutagenic DNA double strand break repair, a pathway implicated in adaptive mutagenesis and antibiotic resistance.
Aim 3 : How do conformational dynamics of Pol III regulate TLS polymerase access and synthesis? In order to determine how Pol IV binds and dissociates from the b2 clamp, we will use single-molecule FRET to follow the conformational dynamics of the polymerase-clamp complex during active replication. Furthermore, we will determine how DNA lesions on the template strand influence these conformational dynamics.
Aim 4 : Identify factors that influence the competition between TLS at the fork and repriming Repriming of DNA synthesis downstream of a lesion competes with TLS to resolve stalled replication forks. We present preliminary cellular and in vitro data that accessory helicases accelerate repriming kinetics. In this aim we will work to identify the helicases that can exert this effect and their mechanism of action.
Chemical damage to DNA occurs frequently and blocks the progression of the replisome, the multiprotein complex responsible for replicating chromosomes. Translesion synthesis is an error-prone DNA damage tolerance pathway that directly bypasses DNA lesions, resolving these dangerous replication blocks. This project uses advanced single-molecule microscopy approaches along with biochemical and genetic assays to determine the mechanism and regulation of translesion synthesis.
|Thrall, Elizabeth S; Kath, James E; Chang, Seungwoo et al. (2017) Single-molecule imaging reveals multiple pathways for the recruitment of translesion polymerases after DNA damage. Nat Commun 8:2170|
|Lee, David F; Lu, Jenny; Chang, Seungwoo et al. (2016) Mapping DNA polymerase errors by single-molecule sequencing. Nucleic Acids Res 44:e118|
|Kath, James E; Chang, Seungwoo; Scotland, Michelle K et al. (2016) Exchange between Escherichia coli polymerases II and III on a processivity clamp. Nucleic Acids Res 44:1681-90|
|Scotland, Michelle K; Heltzel, Justin M H; Kath, James E et al. (2015) A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis. PLoS Genet 11:e1005507|