Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Enzyme dynamics during lesion bypass at the E. coli  replication fork  (#109)

Gurleen Kaur 1 2 , Jacob Lewis 1 2 , Lisanne Spenkelink 1 2 , Slobodan Jergic 1 2 , Nicholas Dixon 1 2 , Antoine van Oijen 1 2
  1. School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia
  2. Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia

While duplicating DNA the replisome encounters a multitude of roadblocks, such as DNA lesions. The replisome needs to be able to efficiently bypass these lesions to ensure replication fidelity and genome stability. Conventional biochemistry methods have been used to study how lesions are bypassed on the template DNA by the replisome [1-3]. The proposed models of lesion bypass do not account for the inherent plasticity of the replisome where proteins can dynamically exchange into the replisome in a manner dependent on their concentration [4, 5]. We aim to study the effect of protein dynamics on lesion bypass using single-molecule fluorescence microscopy.

Currently there are no suitable DNA substrates containing site-specific lesions suitable to study Escherichia coli DNA replication. To determine the molecular details of replisome bypass of template DNA lesions at the single-molecule level, a linear DNA template containing site-specific lesions is required. We have designed and constructed a modular linear DNA substrate containing a site-specific lesion that is readily visualized with single-molecule resolution. Moreover, using E. coli replisomes reconstituted from purified proteins we have observed rates of replication on this template consistent with previous studies. By using fluorescently labeled polymerases, we can simultaneously visualize DNA replication and polymerase dynamics upon lesion collision. For the first time, this single-molecule assay allows for investigation of the interplay between leading-strand lesion bypass and polymerase exchange dynamics.

References

  1. D. Rupp, P. Howard-Flanders, 1968, ‘Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation’, J. Mol. Biol. 31, 291–304.
  2. T. Yeeles, K. J. Marians, 2011 ‘The Escherichia coli replisome is inherently DNA damage tolerant’, Science, 334, 235–238.
  3. T. Yeeles, K. J. Marians, 2013, ‘Dynamics of leading-strand lesion skipping by the replisome’, Mol. Cell, 52, 855–865.
  4. Beattie, T.R., Kapadia, N., Nicolas, E., Uphoff, S., Wollman, A.J., Leake, M.C. and Reyes-Lamothe, R., 2017, ‘Frequent exchange of the DNA polymerase during bacterial chromosome replication’, eLife, 6, e21763.
  5. Lewis, J.S., Spenkelink, L.M., Jergic, S., Wood, E.A., Monachino, E., Horan, N.P., Duderstadt, K.E., Cox, M.M., Robinson, A., Dixon, N.E., and van Oijen, A.M., 2017, ‘Single-molecule visualization of fast polymerase turnover in the bacterial replisome,’ eLife, 6, e23932.