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Real-Time Dynamics of Mutagenesis Reveal the Chronology of DNA Repair and Damage Tolerance Responses in Single Cells

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Real-Time Dynamics of Mutagenesis Reveal the Chronology of DNA Repair and Damage Tolerance Responses in Single Cells Real-time dynamics of mutagenesis reveal the chronology of DNA repair and damage tolerance responses in single cells Stephan Uphoffa,1 aDepartment of Biochemistry, University of Oxford, OX1 3QU Oxford, United Kingdom Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved May 23, 2018 (received for review January 21, 2018) Evolutionary processes are driven by diverse molecular mecha- expression of direct repair (DR) and base excision repair (BER) nisms that act in the creation and prevention of mutations. It pathways to remove alkylation lesions (12, 13). Constitutively remains unclear how these mechanisms are regulated because expressed DR and BER genes complement the inducible genes of limitations of existing mutation assays have precluded measuring the adaptive response. In contrast, DNA damage tolerance via how mutation rates vary over time in single cells. Toward this goal, I translesion synthesis (TLS) or homologous recombination (HR) detected nascent DNA mismatches as a proxy for mutagenesis and enables replication forks to bypass alkylation lesions without re- – simultaneously followed gene expression dynamics in single Escher- pair (14 16). Bacteria control DNA damage tolerance pathways ichia coli through the SOS response, a large gene network that is induced by cells using microfluidics. This general microscopy-based – approach revealed the real-time dynamics of mutagenesis in re- DNA breaks or stalled replication forks (17 19). Whereas Ada- sponse to DNA alkylation damage and antibiotic treatments. It also regulated DR and BER pathways accurately restore the original enabled relating the creation of DNA mismatches to the chronology DNA sequence, SOS-regulated TLS polymerases are intrinsically error prone (16), but error-free lesion bypass and replication re- of the underlying molecular processes. By avoiding population av- start mechanisms are also activated by the SOS response (14, 15). eraging, I discovered cell-to-cell variation in mutagenesis that corre- DNA mismatch repair (MMR) corrects most misincorporated lated with heterogeneity in the expression of alternative responses bases and short insertion/deletion loops before they turn into GENETICS to DNA damage. Pulses of mutagenesis are shown to arise from stable mutations (20, 21). transient DNA repair deficiency. Constitutive expression of DNA re- Despite extensive characterization of the individual DNA re- pair pathways and induction of damage tolerance by the SOS re- pair and damage tolerance pathways, it is still unclear how their sponse compensate for delays in the activation of inducible DNA overlapping and counteracting functions collectively control repair mechanisms, together providing robustness against the toxic mutation rates. Furthermore, DNA damage responses change and mutagenic effects of DNA alkylation damage. the expression and activity of the pathways. How do these dy- namics influence mutation rates? These questions are unsolved mutagenesis | DNA repair | gene expression noise | antibiotics | due to limitations of existing methods to measure mutagenesis. single-cell microscopy Firstly, in vitro biochemical experiments or bulk genetics assays do not reveal the dynamics of DNA repair and mutagenesis in NA damaging agents are widely used as antibiotics and living wild-type cells. Secondly, DNA sequencing or fluctuation Dcancer therapy drugs. These include DNA alkylating, oxidiz- tests provide population- or time-averaged mutation rates from ing, and cross-linking agents, and inhibitors of DNA transactions which the underlying molecular mechanisms can only be inferred (1–3). However, besides the intended cytotoxicity, DNA damage also leads to heritable mutations that can accelerate disease pro- Significance gression and cause drug resistance in pathogenic bacteria and cancers (4–8). In addition, drug treatments trigger cellular stress A central goal in genetics is to understand how mutation rates responses that actively generate mutations (9). The molecular are regulated by the genome maintenance system in response to mechanisms of mutagenesis during normal cell growth and in re- DNA damage or drug treatments. This has been challenging be- sponse to DNA damage have been the focus of intense research cause existing mutation assays only show time and population and debate for decades. Owing to these efforts, many genes have averages of mutation rates and do not resolve the underlying been identified that affect mutation rates, as well as regulatory molecular processes. Toward this goal, I utilized a microscopy- mechanisms that control their expression. However, we lack a clear based method which enables relating the creation of DNA mis- understanding of how mutation rates are defined by the action of matches to single-cell gene expression dynamics in real time. I the replication and repair machinery as a whole. Which factors show that DNA alkylation damage causes a distinct pulse of determine whether a mutagenic DNA lesion is accurately repaired mutagenesis that is shaped by the chronology of constitutive and or converted into a mutation? To address these unknowns, new inducible DNA repair and damage tolerance pathways. Stochastic experimental approaches are required that can measure the real- fluctuations in the expression of these pathways modulated the time dynamics of repair and mutagenesis in a way that individual dynamics of mutagenesis in single Escherichia coli cells. mutation events can be linked to the underlying molecular pro- Author contributions: S.U. designed research, performed research, contributed new re- cesses in live cells. agents/analytic tools, analyzed data, and wrote the paper. Faithful completion of DNA replication is crucial for cell survival and genome stability. Therefore, multiple highly con- The author declares no conflict of interest. served mechanisms that deal with DNA damages exist in all do- This article is a PNAS Direct Submission. mains of life from bacteria to humans (10). These mechanisms fall Published under the PNAS license. broadly into two categories: damage repair and damage tolerance. Data deposition: The data reported in this paper have been deposited in the Oxford An abundant type of DNA damaging agents in the environment University Research Archive, https://doi.org/10.5287/bodleian:Y5R1z6Ev7. and inside cells are alkylating chemicals, which form base lesions 1Email: [email protected]. that perturb the progression and fidelity of DNA synthesis (11). In This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Escherichia coli and many other diverged bacteria, the adaptive 1073/pnas.1801101115/-/DCSupplemental. (Ada) response senses DNA alkylation damage and induces the www.pnas.org/cgi/doi/10.1073/pnas.1801101115 PNAS Latest Articles | 1of10 Downloaded by guest on September 24, 2021 indirectly (20–22). Thirdly, it has become evident that the expression Results of DNA repair proteins is heterogeneous within cell pop- Real-Time Measurements of DNA Mismatch Rates. The action of a ulations (23–25), which may cause cell-to-cell variation in mu- mutagenic process causes DNA mismatches or insertion/deletion tation rates (24, 26–29). This demands experiments with single- loops which can be visualized as foci in live E. coli cells using a cell resolution. Resolving the full distribution of mutation rates fluorescent fusion of the MMR protein MutL-mYPet (Fig. 1A) within a cell population is important for understanding evolutionary (30). Because these foci contain multiple copies of MutL (31), processes, which could be driven not by the population average they are sufficiently bright for detection in standard fluorescence but by a small fraction of individuals in the tail of a distribution microscopy snapshots (SI Appendix, Fig. S1). The frequency of of mutation rates. MMR foci correlates with average genomic mutation rates, and Here, I utilized a general microscopy-based method using can therefore be used as a reporter for mutagenesis (30, 32). microfluidics that complements existing mutation assays. It ad- Automated cell segmentation and tracking was facilitated by dresses the aforementioned challenges by simultaneously moni- constitutive expression of a cytoplasmic mKate2 fluorescent toring the occurrence of DNA mismatches, the expression of protein. To image single cells in a controlled environment, I used DNA damage responses, and the survival of single cells. The a microfluidic device where cells grow inside channels with a formation of MMR foci marks nascent DNA mismatches in real generation time of 42 min under continuous growth medium time, thus providing a direct readout for the activity of mutagenic supply (33) (Fig. 1B). Cell progeny are pushed out of the chan- processes inside cells. This approach revealed a distinct pulse of nels and flow into the waste stream while an individual “mother” mutagenesis in response to constant alkylation damage treatment. cell remains at the end of each channel for long-term observation To determine which factors shape the dynamics, I performed a under constant conditions. Single-cell imaging also avoids any series of genetic perturbations combined with gene expression selection biases that complicate population-based mutagenesis reporters. I discovered a temporal order in the activity of consti- assays. Furthermore, by measuring DNA mismatches instead of tutive and inducible DNA repair and DNA damage tolerance mutations, this approach directly shows the activity of mutagenic pathways, a chronology
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