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Dynamics of the DNA Damage Response: Insights from Live-Cell Imaging Ketki Karanam, Alexander Loewer and Galit Lahav

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Dynamics of the DNA Damage Response: Insights from Live-Cell Imaging Ketki Karanam, Alexander Loewer and Galit Lahav Briefings in Functional Genomics Advance Access published January 4, 2013 BRIEFINGS IN FUNCTIONAL GENOMICS. page 1 of 9 doi:10.1093/bfgp/els059 Dynamics of the DNA damage response: insights from live-cell imaging Ketki Karanam, Alexander Loewer and Galit Lahav Abstract All organisms have to safeguard the integrity of their genome to prevent malfunctioning and oncogenic transform- ation. Sophisticated DNA damage response mechanisms have evolved to detect and repair genomic lesions. With the emergence of live-cell microscopy of individual cells, we now begin to appreciate the complex spatiotemporal kinetics of the DNA damage response and can address the causes and consequences of the heterogeneity in the responses of genetically identical cells. Here, we highlight key discoveries where live-cell imaging has provided unpre- cedented insights into how cells respond to DNA double-strand breaks and discuss the main challenges and promises in using this technique. Downloaded from Keywords: live-cell imaging; single cell; DNA damage; fluorescence microscopy; dynamics INTRODUCTION Modern medicine has exploited the DDR for The integrity of our genome is continually chal- cancer treatment [6]. Frontline therapies, such as http://bfg.oxfordjournals.org/ lenged by damaging agents such as reactive bypro- radiation and chemotherapy, function by generating ducts of cellular metabolism, background radiation high levels of DNA damage, often in the form of and environmental mutagens. Of the various types DSBs. Cancer cells are more susceptible to the of DNA lesions that arise within the cell, DNA adverse effects of DNA damage compared with double-strand breaks (DSBs) are particularly danger- healthy cells, as they frequently harbor mutations ous as their improper repair can cause chromosomal that compromise the DDR and cell cycle check- rearrangements that promote oncogenic transform- points. However, the effectiveness of anti-cancer by guest on January 10, 2013 ation [1, 2]. DSBs activate a complex DNA damage therapies depends on the repair capacity of the response (DDR) cascade, which includes recognition targeted cells, highlighting the importance of under- of the damage and initiation of repair, as well as standing the mechanisms and dynamics of the DDR amplification of the damage signal and its transmis- in healthy and transformed cells. sion to checkpoints that temporarily halt the cell A characteristic outcome in cancer therapy is cycle and allow additional time for repair [3]. ‘fractional killing’, where each round of therapy In case full repair is not achieved, the DDR directs kills some but not all cells in a tumor [7]. damaged cells towards permanent cell cycle arrest Traditionally, this was thought to result from differ- or apoptosis. A functional DDR is therefore vital ences in the cells’ accessibility and sensitivity to the for preserving genomic integrity and many features drug, the involvement of cancer stem cells or from of this response are highly conserved from yeast to genetic heterogeneity between cells of the same humans [4, 5]. tumor [8–10]. However, much recent work has Corresponding author. Galit Lahav. Tel.: 617-432-5621; Fax: 617-432-5012; E-mail: [email protected] Ketki Karanam is a PhD candidate at the Department of Systems Biology at Harvard Medical School (Boston, MA, USA). She uses quantitative live-cell imaging to study DNA double-strand break repair in single human cells. Alexander Loewer is a group leader at the Max Delbruek Center (Berlin, Germany). His group uses live-cell time-lapse microscopy to understand signaling dynamics in single cells, focusing on the interplay of cellular networks. Galit Lahav is an associate professor at the Department of Systems Biology at Harvard Medical School (Boston, MA, USA). Her group combines experimental and theoretical approaches to study the dynamics of signaling networks and cellular decision-making in single cells. ß The Author 2013. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected] page 2 of 9 Karanam et al. shown a large cell-to-cell variability in phenotypes pooled DNA from a large number of damaged cells and behaviors of genetically identical cells even when [16]. Single-cell methods such as the comet assay they are grown in the same conditions and treated allowed detection of the levels of DNA damage in uniformly [11, 12]. Such cellular heterogeneity individual cells; however, it was difficult to relate among isogenic cells was previously shown to have these measurements to the exact number of breaks implications in numerous cellular processes including in a cell [17]. In addition, both methods investigated the activation of T cells [13], the timing of apoptosis DNA repair outside the normal physiological context [14] and the differentiation of stem cells [15]. (i.e. outside the nucleus of a cell). The subsequent Similarly, it is possible that the incomplete killing discovery that DNA repair proteins and modified his- of tumors in response to DNA-damaging drugs re- tones organize into macromolecular structures (foci) sults from differences in the DDR of individual around break sites, which can be easily visualized by tumor cells arising from stochastic fluctuations in immunofluorescence and light microscopy, provided gene expression and protein levels, differences in cel- a breakthrough advance in our ability to observe the lular states (such as cell cycle phase) or an altered number and location of DSBs in situ, in single cells microenvironment. In order to gain deeper insights [18–21]. Such analyses have been instrumental in pro- into factors that regulate the DDR and to accurately viding key conceptual advances regarding the identity predict the cellular response to DNA damage, it is and function of various factors involved in the DDR Downloaded from therefore essential to observe these responses at the and DSB repair [22–25]. single-cell level. While the visualization of damaged-induced foci Historically, DNA repair has been investigated by immunofluorescence allowed quantification of using molecular and biochemical approaches that distinct DSBs in the nucleus, the ‘kinetics’ of the are based on measurements of populations of cells. repair process was inferred by averaging across popu- http://bfg.oxfordjournals.org/ For example, early measurements of the kinetics of lations of cells fixed at specific times after damage. DSB repair were obtained from physical estimates of Such measurements may not be representative of the DNA size using pulsed-field gel electrophoresis that true kinetics in single cells (Figure 1). Moreover, cell by guest on January 10, 2013 Figure 1: Ensemble measurements can mask the variation in the kinetics of repair between cells. The kinetics of DNA DSB repair are popularly measured by averaging the number of foci in groups of fixed cells at specific times after damage. Such measurements do not capture the natural variation in the population and can mask the true kin- etics of repair in single cells. (A) Leftça homogeneous population that repairs rather uniformly and rightça hetero- geneous population that exhibits bimodality in the rates of repair; both yield the same average measurements at different times after damage. (B) The decay in the number of foci for three different cells is shown. Each of these cells has unique kinetics of repair, however, would yield the same number of foci if fixed at the indicated times. Live-cell imaging of DDR page 3 of 9 fixation precludes insight into the precise dynamics The rate at which the fluorescence recovers provides and order of events at the sites of DNA damage. a measure of the mobility of the tagged protein, Live-cell imaging has recently emerged as a powerful which is determined by its rate of diffusion and its technique for visualizing and quantifying the DNA in vivo binding interactions. repair process over time in single cells. This tech- To assess if the mobility of the fusion protein is nique has been elegantly utilized for understanding influenced by interactions with other proteins or cel- the spatiotemporal aspects of the DDR such as the lular compartments, its FRAP recovery is compared mobility of repair proteins [26, 27] and their assem- with that of an inert, non-binding molecule, ideally bly into foci at damage sites [28–30], as well as the of the same size. When the tagged protein has a movements of damaged DNA and chromatin slower FRAP recovery than the inert counterpart, [31–39]. In the following sections, we describe transient binding is implicated and the degree of these key areas in which live-cell imaging has pro- slowdown in the FRAP recovery provides a measure vided unprecedented insights into the DDR and of binding kinetics. Furthermore, the proportion of outline the main experimental techniques used for the original fluorescence that is recovered over the these analyses. We also discuss the limitations of measurement time indicates the mobile protein frac- using live-cell reporters for studying cellular re- tion in the bleached region. When the mobile frac- sponses and conclude with open questions that tion is less than 100%, some fluorescent molecules Downloaded from have the potential to be addressed using live-cell may be stably bound to a fixed substrate such as a imaging methods. DSB site. In FLIP, one region of the cell is photo- bleached, and the loss of fluorescence is recorded in another region of the cell (Figure 2B). Different spe- http://bfg.oxfordjournals.org/ INSIGHTS FROM SHORT-TERM cies of the tagged protein migrate into the bleached LIVE-CELL IMAGING OF THE DDR region at different rates; hence, FLIP provides meas- Imaging DNA repair, and more generally biological urements of the steady state exchange of proteins processes, in living cells has been revolutionized by between the two compartments as well as the bind- the discovery of green fluorescent protein (GFP) and ing status of the tagged protein and the relative pro- the subsequent development of its spectral variants portions of its different states [59].
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