The characterized microirradiation system LSM700 can now be used as a suitable tool to study DNA repair factors by live-cell monitoring or after DNA damage induction in fixed cells by immunocytochemistry.
Fluorescence labeling of regulator proteins of DNA repair grants information about their localization changes or recruitment kinetics upon DNA damage.
7.4.1 Polymorphism-dependent recruitment of PARP1 and the corresponding physiological consequences
Poly(ADP-ribose) polymerase 1 (PARP1) is tightly involved in different cellular processes and regulates the synthesis of the posttranslational modification PAR (Schreiber, Dantzer et al. 2006). Upon DNA base modifications and single-strands breaks PARP1 is activated by autoPARylation and modifies other repair factors with PAR leading to the recruitment to damaged sites and triggering repair processes.
Knockout studies in mice and siRNA-mediated knockdown studies in cells have demonstrated that the loss of PARP1 leads to embryonic lethality and existing PARP1 polymorphisms are linked to an increased risk of cancer development (Shall and de Murcia 2000, Blenn, Wyrsch et al. 2012). In turn, it was shown that PARP1 is overexpressed in different types of cancer (Swindall, Stanley et al. 2013).
In this study, TALEN-mediated HeLa PARP1 knockout cells were reconstituted with different eGFP-PARP1 variants to analyze their recruitment to sites of laser-induced DNA damage. The naturally occurring polymorphisms, V762A and F304L, are variants with mutations in the enzymatically active center of the protein (Yu, Ma et al. 2012). A significant decrease of the maximum recruitment level of these variants may support the hypothesis of a reduced PARP1 activity resulting in an increased risk of cancer development based on genomic instability (Rank, Veith et al. 2016).
In contrast to the two naturally occurring PARP1 variants, artificially generated PARP1 mutants were studied as well. The mutant L713F was previously described as a gain-of-function hypermorphic mutant with an increased level of active PARP1 (Miranda, Dantzer et al. 1995). Recruitment analysis of this variant supports the generation of the gain-of-function mutant by a significant increase of the maximum recruitment level, but similar dissociation kinetics. Quite contrary to the described hypermorphic L713F variant, Mortusewicz et al. developed a hypomorphic variant (E988K), which incorporates a mutation within the catalytic triad. This mutation leads to a strongly reduced PARP1 activity resulting only in mono(ADP-ribos)ylation of target proteins (Mortusewicz, Ame et al. 2007). The performed recruitment analysis of PARP1 E988K showed a delayed accumulation and a longer persistence at sites of laser-induced DNA damage. These findings are in line with the study of Mortusewicz et al. identifying that the catalytic activity is not only responsible for targeting the DNA damage, but also for the dissociation from damaged sites.
The additional transfection of an RFP-tagged XRCC1 demonstrated physiological consequences of an impaired PARP1 recruitment. In the presence of the catalytic inactive PARP1 variant E988K, no recruitment of XRCC1-RFP could be observed.
Supported by findings of Li et al., this result exhibit that the recruitment of subsequent repair proteins is mediated by PAR chains, but mono(ADP-ribos)ylation might not be sufficient for further signaling (Li and Yu 2013).
In general, our microirradiation setup is a suitable tool for studying the recruitment behavior of PARP1 and its variants. Live-cell monitoring of fluorescently tagged PARP1 variants offers information about localization changes upon damage induction. Using a
HeLa cell line with a TALEN-mediated PARP1 knockout enables the analysis of different protein variants in an easy, fast and reproducible manner.
7.4.1.1 Negative influence of double transfection on recruitment
During recruitment experiments of double transfected cells (PARP1-GFP and XRCC1-RFP), it could be observed that the maximum recruitment level of PARP1 in the presence of XRCC1-RFP is significantly lower than the recruitment level in experiments without XRCC1-RFP. This might be explained by the size of the fluorophore-tagged proteins and the subsequent destruction of the fluorescence signal by quenching processes.
PARP1 and XRCC1 are classified as strong recruiters to sites of laser-induced DNA damage (Polo and Jackson 2011). In this study, both of them are overexpressed and additionally labeled with fluorescent proteins. Upon damage induction, PARP1 recruits to damaged sites and mediates the recruitment of XRCC1 for further repair processes.
Concerning the available small volume after non-linear damage induction and the size of the two fluorophores, the strong recruitment of these proteins may result in steric inhibition demonstrated in a reduced efficiency of the process compared to the single transfection. Additionally, the massive recruitment of both proteins may lead to quenching processes of their fluorophores with PARP1-GFP as donor and XRCC1-RFP as acceptor protein. GFP and RFP as fluorescence labels present overlapping emission and excitation spectra. In quenching processes, the emitting GFP signal is simultaneously used as excitation for the RFP fluorophore leading to a reduction in the GFP signal (Day and Davidson 2012).
7.4.2 Real-time imaging of PARylation
As mentioned above, upon DNA damage PARP1 is activated and further regulates the effective recruitment of repair proteins to the damaged sites by modifying them with PAR.
In this study, the PAR turnover could be monitored in living cells, achieved by a successful tagging of its component NAD+ with the fluorescent dye TMR (Wallrodt, Buntz et al. 2016). To investigate the biological functionality of this 2TMR-NAD+ molecule, localized DNA damage was induced using the microirradiation system. It could be quantitatively shown that the fluorescently labeled 2TMR-NAD+ accumulates at laser-induced DNA damage. Upon treatment with the PARP1 inhibitor ABT-888, no signal accumulation could be observed indicating that the effect is completely dependent on PARP1/2 activity.
As described in the previous sections, PAR chains have the ability to mediate the recruitment of other factors for coordinating an efficient repair process (Tallis, Morra et al. 2014). One of the known PAR binding domains is the macrodomain, which represents the non-histone like C-terminus of the histone variant macroH2A (Angelov, Molla et al.
2003). It binds PAR either covalently or non-covalently (Karras, Kustatscher et al. 2005).
Here, the additional transfection of macroH2A-GFP in 2TMR-NAD+ treated cells was used to test simultaneously the accumulation of 2TMR-NAD+ and the subsequent
recruitment of macroH2A-GFP to sites of laser-induced DNA damage. According to the PAR turnover, the signal of 2TMR-NAD+ first accumulates and then decreases over time, while the dissociation of macroH2A-GFP was delayed indicating a longer persistence at damaged sites. Further evaluation of this effect revealed that the less efficient recruitment and the longer persistence of macroH2A-GFP are caused by the treatment with the protein carrier Pep1. This amphipathic carrier peptide was used to facilitate the cellular uptake of the negatively charged 2TMR-NAD+. It consists of a hydrophobic domain, which is responsible for interactions with proteins and the cell membrane. A hydrophilic domain ensures intracellular delivery and solubility (Morris, Depollier et al. 2001, Henriques and Castanho 2004). In macroH2A-GFP transfected cells, but without the presence of 2TMR-NAD+, Pep1 might interact with macroH2A via its hydrophobic properties. Because of the massive recruitment to sites of laser-induced DNA damage, Pep1 might not only interact with the protein but may influence its recruitment behavior. Through its hydrophobic interactions, Pep1 might stabilize the recruited macroH2A-GFP in the complex of the huge repair machinery resulting in a longer persistence at sites of DNA damage.