The Reyes Lab aims to understand the function of molecular machines as they work inside the cell
Our group uses single-molecule approaches in live cells to infer the spatial organization and determine dynamics of proteins, to study the movement and rate of synthesis of DNA and to determine the kinetics of cellular processes. Our current focus is in the understanding of the molecular machines of DNA replication. We use the unicellular organisms, E. coli and S. cerevisiae, as model organisms for bacteria and eukaryotes.
Spying the dynamics inside the replisome
Outline of E.coli with a fluorescent focus representing a single molecule of mMaple fused to the DnaB helicase. Pictures taken every 2 minutes.
The replisome is composed of multiple different proteins, some of them present in more than one copy per replication fork. Once assembled, the bacterial replisome is able to copy millions of bases pairs. In contrast with this stability, cycles of binding and unbinding of individual subunits define how DNA replication is regulated at initiation, how it achieves periodic cycles of synthesis at the lagging strand, how does it prevent premature termination, how the replisome bypasses lesions on DNA, and how does replication terminates. There are still many gaps in our understanding on the dynamics inside the replisome. Our ignorance is partly due to the difficulty of determining the fate of individual components of the replisome while actively synthesising DNA.
By focusing sensitive microscopes on cells undergoing DNA replication, we use quantitative approaches to determine how often does the replisome replace its components during DNA replication. This information will allow us to understand how DNA replication is carried out and what factors determine the stability of replisome as a protein assembly. We are also interested in observing the recruitment and unbinding of components at initiation and termination, in order to better understand these processes.
Defining the eukaryotic replisome in live cells
Outline of two S. cerevisiae cells labelled with PCNA-mNeonGreen (left), one of them undergoing S-phase. A close-up view of a Z-projection of the replicating nucleus obtained by Structural Illumination (right). Multiple fluorescent foci representing each one or more replisomes are observed.
It has been only in the last decade that we have finish identifying (probably) all the subunits of the replisome in yeast. Great efforts in the community are now focused in understanding how these components assemble into a functional machine. Our lab aims at contributing to understand the architecture of the replisome, the inner dynamics in the replisome, and how its coordinated function results in the synthesis of the chromosome. Achieving a high signal-to-noise ratio in fluorescence microscopy for quantitative approaches is more complicated than in bacteria due to higher levels of endogenous and out-of-focus fluorescence in yeast. Our lab is developing new microscopy, genetic and labelling approaches that allows the study of live yeast using single-molecule and super-resolution microscopy techniques.
In addition to the understanding of the replisome, we are also working on the mechanisms that regulate origin firing during S-phase. S. cerevisiae has hundreds of potential origins, many of which fire at different stages of S-phase. Origin selection is established by multiple factors that include DNA sequence, chromatin factors and the action of limiting initiation factors. We are currently working to determine the distribution in the genome and the kinetics of initiation factors.
Directed evolution of DNA Polymerase
Cells expressing mutant forms of DNA Pol III are trapped in bubbles of water-in-oil emulsion containing PCR mix (left). Outline of two bubbles containing a single cell each (middle). Three different views of the palm domain's structure of Pol III (right).
Our lab constantly searches for new tools to stimulate controlled responses from cells that can help us understand their biochemistry. An important point of control in our studies is the replicative polymerase, DNA Pol III, which is in charge of synthesizing the chromosome. Pol III has evolved to have very high fidelity at incorporating nucleotide residues during the elongation of DNA. In good measure, this is due to high efficiency at discriminating between substrate nucleotides. However, in the lab this substrate specificity limits our capacity to track the Pol III activity in cells. Bulky base analogues, like those coupled to a fluorescent dye, will not be incorporated into DNA, even when the base they carry is one of the four natural bases for DNA. In order to obtain new ways to track and manipulate Pol III activity, we are using directed evolution techniques to generate Pol III mutants that maintain high fidelity of incorporation but relaxed or different nucleotide selectivity
The cell biology of antibiotic resistance
E. coli cells carrying the plasmid pJHCMW1 that codes for multiple genes of antibiotic resistance. The plasmid is shown in green (labelled by tetO array/TetR-Ypet system) and the chromosome is shown in red.
In collaboration with the group of Marcelo Tolmasky at CSU Fullerton, we are studying how antibiotic resistance is established in the bacterial cell. In recent published work we determined the distribution of a multi-resistance plasmid in cells, showing that plasmids preferentially localize at the edges of the cell, where there is a low density of chromosomal DNA (Reyes-Lamothe et al, 2014). We are currently using fluorescence microscopy to measure the activity of the antibiotic resistance enzymes in live cells. We aim to establish enzymatic kinetics in vivo and determine the cellular factors that influences it.
- Canada Research Chair in Chromosome Biology
- D.Phil. (University of Oxford)
- M.Sc. (Concordia University)
- B.Sc. (UNAM, Mexico)
- Ph.D. (University of Edinburgh)
- Ziad currently studies the initiation of replication in yeast and the mechanisms of substrate fidelity in the bacterial PolIII.
- Ph.D.(Universite de Montreal)
- Maxime works on initiation and re-initiation of DNA replication in E. coli
- B.Sc. (University of Toronto)
- Nitin works to develop tools for the study of yeast using single-molecule microscopy.
- B.Sc. (Sacred Heart University)
- Nic studies fidelity in the bacterial replicative polymerase with the aim of exploiting this knowledge for the study of DNA replication.
- B.Sc. (McGill University)
- Angela currently studies the initiation of DNA replication in yeast using super-resolution microscopy.
- B.Sc.(McGill University)
- Mostafa studies the activity of primase in E. coli
Major Biology and Mathematics (McGill)
- Angela aims to simulate microscopy data to improve our protocols for data acquisition and analysis
- Fong Hue, Visiting Masters Student Sumers of 2015 & 2016 (Currently: Graduate student at the California State University, Fullerton, USA)
- Florian Commans, Visiting Masters Student Winter-Summer 2016 (Currently: Travelling around the globe)
- Yin Xin Ho, Undergraduate Student 2015-2016 (Currently: Graduate student at the University of Manchester, UK)
- Thomas Beattie, Postdoctoral Fellow 2013-2016 (Currently: Health Information Officer, Breast Cancer Now, UK)
- Andrea Wang, Undergraduate Student 2015-2016 (Currently: Student at McGill University)
- Michael Reaume, Undergraduate Student Winter 2016 (Currently: Student at McGill University)
- Vivi Ma, Graduate Student 2013-2016
- Jia Lu (Angela), Undergraduate Student 2015-2016 (Currently: Student at McGill University)
- Tuo Yu (Stella), Undergraduate Student Summer 2015 (Currently: Student at McGill University)
- Xiaoyu Wei, Undergraduate Student Winter 2015
- Harsh Aurora, Undergraduate Student Winter 2014-2015 (Currently: Graduate student at McGill University)
- Alina Phen, Undergraduate Student Fall 2014 (Currently: Dental School, University of Toronto)
- Chloe Pou-Prom, Undergraduate Student Summer 2014 (Currently: Graduate Student at the University of Toronto)
- Beattie T, Reyes-Lamothe R (2015) Replisome’s journey through the bacterial chromosome. Frontiers in Microbiology 6: 562. doi: 10.3389/fmicb.2015.00562
- Moolman, M. C., Krishnan S. T., Kerssemakers J. W., van den Berg A., Tulinski P., Depken M., Reyes-Lamothe R., Sherratt D. J. and Dekker N. H. (2014) Slow unloading leads to DNA-bound beta2-sliding clamp accumulation in live Escherichia coli cells. Nat Commun 5: 5820.
- Reyes-Lamothe R, Tran T, Meas D, Lee L, Li AM, Sherratt DJ, Tolmasky ME (2014). High-copy bacterial plasmids diffuse in the nucleoid-free space, replicate stochastically and are randomly partitioned at cell division. Nucleic Acids Res. 42, 1042-51.
- El-Hajj ZW, Reyes-Lamothe R, & Newman EB (2013). Cell division, one-carbon metabolism and methionine synthesis in a metK-deficient Escherichia coli mutant, and a role for MmuM. Microbiology 159, 2036-2048.
- Uphoff, S., Reyes-Lamothe, R., Garza de Leon, F., Sherratt, D.J., Kapanidis, A.N. (2013). Single-molecule DNA repair in live bacteria. Proc Natl Acad Sci U S A 110, 8063-8068.
- Badrinarayanan, A., Reyes-Lamothe, R., Uphoff, S., Leake, M.C., and Sherratt, D.J. (2012). In vivo architecture and action of bacterial structural maintenance of chromosome proteins. Science 338, 528-531.
- Reyes-Lamothe, R., Nicolas, E., and Sherratt, D.J. (2012). Chromosome Replication and Segregation in Bacteria. Annu Rev Genet 46, 121-143.
- Badrinarayanan, A., Lesterlin, C., Reyes-Lamothe, R., and Sherratt, D. (2012). The Escherichia coli SMC Complex, MukBEF, Shapes Nucleoid Organization Independently of DNA Replication. J Bacteriol 194, 4669-4676.
- Tran, T., Andres, P., Petroni, A., Soler-Bistue, A., Albornoz, E., Zorreguieta, A., Reyes-Lamothe, R., Sherratt, D.J., Corso, A., and Tolmasky, M.E. (2012). Small plasmids harboring qnrB19: a model for plasmid evolution mediated by site-specific recombination at oriT and Xer sites. Antimicrobial agents and chemotherapy 56, 1821-1827.
- Reyes-Lamothe, R. (2012). Use of Fluorescently Tagged SSB Proteins in In Vivo Localization Experiments. Methods Mol Biol 922, 245-253.
- Wang, X., Lesterlin, C., Reyes-Lamothe, R., Ball, G., and Sherratt, D.J. (2011). Replication and segregation of an Escherichia coli chromosome with two replication origins. Proc Natl Acad Sci U S A 108, E243-250.
- Gonzalez-Silva, N., Lopez-Lara, I.M., Reyes-Lamothe, R., Taylor, A.M., Sumpton, D., Thomas-Oates, J., and Geiger, O. (2011). The dioxygenase-encoding olsD gene from Burkholderia cenocepacia causes the hydroxylation of the amide-linked fatty acyl moiety of ornithine-containing membrane lipids. Biochemistry 50, 6396-6408.
- Reyes-Lamothe, R., Sherratt, D.J., and Leake, M.C. (2010). Stoichiometry and architecture of active DNA replication machinery in Escherichia coli. Science 328, 498-501.
We look for highly motivated students with biology, physics, computational or chemistry backgrounds aiming to enroll in a MSc or PhD program. Enquiries with CV and one-page research proposal are welcome.
Dr. Rodrigo Reyes-Lamothe
Department of Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
Tel: (514) 398-5137 Fax: (514) 398-5069