DNA replication and replication termination in ... DNA replication and replication termination in Escherichia

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  • DNA replication and replication termination

    in Escherichia coli

    A thesis submitted

    for the degree of Doctor of Philosophy

    by Sarah Midgley-Smith

    Department of Biosciences,

    Brunel University London

    October 2018

  • 2

    Abstract

    A prerequisite for successful cell division is the generation of an accurate copy of the entire genome

    as well as faithful segregation into the daughter cells. In the bacterium Escherichia coli, replication of

    the circular chromosome is initiated at a single origin (oriC) where two replication forks are

    assembled and proceed bi-directionally until they converge within a defined termination region

    opposite oriC and fork fusion takes place. This region is flanked by ter sequences, which, when bound

    by Tus protein, form a replication fork trap that allows forks to enter but not to leave. While the events

    associated with initiation as well as the elongation of replication have been extensively studied, the

    molecular details associated with the fusion of two replisomes are far less well characterised.

    The data presented here significantly extend our understanding of the molecular mechanics

    associated with the fusion of two replisomes in E. coli. My results strongly support the idea that RecG

    is a key player in processing intermediates that arise as two replication forks fuse. In the absence of

    RecG, over-replication of the chromosome is initiated at fork fusion intermediates, a process that can

    take place outside of the native termination area if forks are forced to fuse in an ectopic location. RecG

    has also been implicated in processing recombination intermediates. Over-replication in the absence

    of RecG is dependent on recombination and my data support the idea that RecG is important in limiting

    replication that initiates at recombination intermediates, some of which arise as a consequence of fork

    fusion events. In contrast, my data do not support the notion that the over-replication of chromosomal

    DNA in cells lacking RecG is in any way triggered by R-loops, as suggested previously in the literature.

    The observation that over-replication is taking place in cells lacking exonuclease I strongly

    supports the idea that 3’ single-stranded DNA structures are a key intermediate of fork fusion events.

    My data demonstrate that 3’ flaps accumulating in the absence of ExoI can be converted into 5’ flaps

    and degraded by 5’ exonucleases such as ExoVII and RecJ. RecG helicase is likely to be involved in this

    conversion. Thus, the data presented in this thesis highlight the complexity of replication fork fusion

    events and demonstrate that multiple protein activities are required to process fork fusion

    intermediates in order to allow DNA replication to be completed with high accuracy and enable

    faithful segregation of complete chromosomes into daughter cells.

  • 3

    Declaration

    The research presented in this thesis is my own work, unless otherwise specified, and has not been

    submitted for any other degree.

    Sarah Midgley-Smith

  • 4

    Acknowledgements

    First and foremost, I would like to thank Dr Christian Rudolph. It has been a privilege and a pleasure

    to work in your lab and I am immensely grateful to you for supporting me in achieving this feat. I will

    miss your enthusiasm, stimulating discussions about our work, and especially your disregard for the

    instruction manual. Thank you for teaching me so much about what it means to be a scientist.

    A great team of people at Brunel have made my time here that much more enjoyable. Thanks to

    the technicians, especially Helen, for always helping out when needed. Special thanks must go to the

    other members of the Rudolph lab, in particular Juachi; I will miss working with you and I am so

    grateful for your kindness and willingness to share your knowledge in the lab. Thanks to some first-

    rate undergrad students, Toni, Monja and Ewa, for producing some great work and ensuring that I was

    never lonely in the lab! Thanks to my fellow PhD students past and present for being an excellent

    source of procrastination; there have been some interesting conversations. Ezgi, I could not have

    wished for a better desk neighbour. Being in the midst of a PhD often felt quite lonely and it was nice

    to know that you were right there with me, sharing successes and (more often) frustration! I am so

    grateful for all your encouragement.

    I could not possibly fail to mention the best friends I can imagine, Louisa, Liz, Tone and Kate. You

    are outstanding. I wish I could see you all every day and I am so lucky to have you as my friends.

    Thanks also to Caroline, Naomi, Pippa, J-Lo and Steph for fab times away from work. Special mention

    to Tommy, for everything.

    To my parents, Dave and Kate; your support has been incredible. Thank you for instilling in me as

    a child the importance of education and always encouraging me to do well, and for being endlessly

    supportive on the many occasions that I have not. I know that you are as pleased about this success as

    I am, and that is very special. I am thrilled to be able to share this with you. Thanks for the well-timed

    Vespers and food that kept me writing through the night!

    My sisters Eleanor and Harriet are second to none. Eleanor, thank you for always being there on

    the end of the phone with all the right things to say to cheer me up; I think the highlight was firework

    cells! Harri, you have provided many welcome distractions from my work, like cooking chilli for 30

    people, or carrying a washing machine up and down stairs! Thank you both for being so fab all the

    times you knew I was not quite on form; you girls are the best.

    Finally, to my husband and my best friend, Greg. I cannot write anything that will do justice to

    what you have done in supporting me through the past 10 years. This success belongs to you as much

    as to me, as the Big Science Story would not have happened without you! You cheer me up and make

    me laugh when I need it most and are always on my side. I am unbelievably lucky to have your constant

    love, help and advice and I know I have not always made it easy. Thank you for being right there next

    to me through all the ups and downs; pub?

  • 5

    Publications

    As a result of my work, several publications have already been published with data that will be

    presented in this thesis, and those publications are:

    Midgley-Smith, S.L.; Dimude, J.U. and Rudolph, C.J. (2019) A role for 3’ exonucleases at the final stages

    of chromosome duplication in Escherichia coli Nucleic Acids Research 47(4):1847-1860

    Dimude, J.U.; Midgley-Smith, S.L. and Rudolph, C.J. (2018) Replication-transcription conflicts trigger

    extensive DNA degradation in Escherichia coli cells lacking RecBCD. DNA Repair (Amst) 70:37–48

    Midgley-Smith, S.L.; Dimude, J.U.; Taylor, T.; Forrester, N.M.; Upton, A.L.; Lloyd, R.G. and Rudolph, C.J.

    (2018) Chromosomal over-replication in Escherichia coli recG cells is triggered by replication fork

    fusion and amplified if replichore symmetry is disturbed. Nucleic Acids Research 46(15):7701-7715

    Dimude, J.U.; Midgley-Smith, S.L.; Stein, M. and Rudolph, C.J. (2016). Replication Termination:

    Containing Fork Fusion-Mediated Pathologies in Escherichia coli. Genes 7(8), 40

    Dimude, J.U.; Stockum, A; Midgley-Smith, S.L.; Upton, A.L.; Foster, H.A.; Khan, A; Saunders, N.J.; Retkute,

    R. and Rudolph, C.J. (2015). The Consequences of Replicating in the Wrong Orientation: Bacterial

    Chromosome Duplication without an Active Replication Origin. mBio. 6(6) e01294-15

    Darja Ivanova, Toni Taylor, Sarah L. Smith, Juachi U. Dimude, Amy L. Upton, Mana M. Mehrjouy, Ole

    Skovgaard, David J. Sherratt, Renata Retkute, Christian J. Rudolph. (2015) Shaping the landscape of

    the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic

    replication origin. Nucleic Acids Research. 43(16): 7865–7877

  • 6

    List of abbreviations

    A600 absorption at 600 nm

    ADP adenosine diphosphate

    amp gene for ampicillin resistance

    apra gene for apramycin resistance

    ATP adenosine triphosphate

    bp base pair

    BrdU bromodeoxyuridine

    cat chloramphenicol acetyl transferase, gene for chloramphenicol resistance

    cfu colony forming units

    cSDR constitutive stable DNA replication

    Dam DNA adenine methyltransferase

    dhfr dihydrofolate reductase, gene for trimethoprim resistance

    dif deletion-induced filamentation, dimer resolution site

    D-loop displacement-loop where the invading species is DNA

    DNA deoxyribonucleic acid

    dNTP deoxynucleoside triphosphate

    DSBR double-strand break repair

    dsDNA double-stranded DNA

    DUE DNA-unwinding element

    EDTA Ethylenediaminetetraacetic acid

    EdU 5-ethynyl-2’-deoxyuridine

    FP fluorescent fusion protein

    frt flippase recognition target

    Hfr high frequency of recombination

    IPTG Isopropyl-β-D-thiogalactoside

    iSDR inducible stable DNA