This project utilizes novel single-molecule approaches to elucidate the mechanisms by which the bacterial replisome regulates access of translesion polymerases to the replication fork and mediates translesion synthesis (TLS) across DNA lesions that block the replisome. Proper regulation of TLS is essential because TLS polymerases are significantly more error-prone than their replicative counterparts. Improper access of TLS polymerases to the replication fork is correlated with increased mutation rates in both prokaryotes and eukaryotes. Answers to fundamental mechanistic questions regarding how TLS occurs remain obscured in ensemble biochemical experiments due to the dynamic nature of TLS polymerase exchange. This project will exploit new developments in single-molecule manipulation and imaging to detect the many structural intermediates of the replisome that transiently arise during translesion synthesis. The three specific aims are:
Aim 1) Determine how the processivity factor mediates polymerase exchange. Processive DNA synthesis by the polymerases of E. coli requires interactions with the bacterial processivity factor, . The clap is believed to be a loading platform for multiple polymerases but how polymerase-clamp interactions mediate polymerase trafficking at the replication fork remains unclear.
This aim will utilize single-molecule approaches to characterize the kinetics of polymerase exchange and elucidate how -polymerase interactions facilitate switching. In parallel, single-molecule imaging of individual fluorescently labeled polymerases will directly quantify polymerase composition and conformation on .
Aim 2) Determine how TLS polymerases associate with the replisome and carry out TLS. TLS is believed to occur either at moving replication forks through polymerase switching reactions or in ssDNA gaps generated by the replisome translocating past the lesion and repriming synthesis downstream. In this aim, single-molecule imaging of fluorescently labeled replisome components in vitro and in cells will be used to determine how TLS polymerases interact with a replisome that has collided with a leading strand lesion.
Aim 3) Identify regulators of TLS within the SOS DNA damage response. Widespread DNA damage leads to induction of the SOS DNA damage response, which results in the transcriptional upregulation of over 40 gene products involved in DNA repair and TLS. On their own, SOS levels of TLS polymerases significantly inhibit replication, leading to suggestions that TLS polymerases may slow replication to allow DNA repair to occur. Yet, strains constitutively active for the SOS response appear to grow normally, indicating that SOS gene products play a role in regulating TLS polymerase access to the fork. We will determine how high concentrations of TLS polymerases remodel the replisome and work to understand how other SOS genes further regulate TLS.

Public Health Relevance

DNA damage occurs frequently, and when left unrepaired, can serve as a potent blockade to the multiprotein machinery responsible for replicating DNA. Left unresolved, these replication blocks can lead to disease-causing mutations or cell death. This project employs novel single-molecule microscopy approaches to visualize how individual translesion polymerases work with the DNA replication machinery to bypass these blocking lesions.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Reddy, Michael K
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Harvard Medical School
Schools of Medicine
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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