DNA polymerase μ is a global player in the repair of non-homologous end-joining substrates
Highlights
► Pol μ is a global player that increases the efficiency and the fidelity of NHEJ. ► Pol μ is implicated in the repair of substrates that do not undergo DNA synthesis. ► DNA synthesis activity of Pol μ is non-essential for NHEJ in a cellular context. ► Diverse repair events are produced according to the type of NHEJ impairment. ► Pol μ-dependent molecular landscapes of NHEJ events in primary cells.
Introduction
Pol μ is a specialized DNA polymerase whose inactivation results in a mild phenotype in vivo, namely in the impairment of a sub-class of immunoglobulin genes rearrangements [1] and in affected haematopoietic development [2]. These effects have been attributed to the implication of pol μ in non-homologous end-joining (NHEJ), a mechanism that repairs DNA double strand breaks (DSBs). DSBs are produced either by the action of a nuclease, as it is the case for immunoglobulin genes, or as a consequence of physiological processes (DNA replication, respiration) or by genotoxic agents. Although non-essential, pol μ contributes to cell fitness by reducing the proliferative decline [2] and senescence [3] of primary fibroblasts, affecting their immortalization after irradiation [3], and maintaining haematopoietic progenitors cells homeostasis [2]. However, the molecular mechanisms by which pol μ intervenes in DSB repair, thereby influencing the cellular response to DNA damage, remain unclear. To date, only limited information is available on how pol μ intervenes in DSB repair in vivo, and this essentially for immunoglobulin gene rearrangements. Intriguingly, in vivo data poorly recapitulate the function of pol μ reported in vitro, and these aspects need to be further elucidated.
The core NHEJ factors Ku, the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and the XRCC4/ligase IV complex, are able to repair compatible ends in vitro [4], stimulated by the XLF/Cernunnos factor [5], [6]. Non-compatible ends are processed by nucleases (including Artemis, and the MRN complex (MRE11/Rad50/NBS1) and DNA polymerases (likely pol λ and pol μ), before ligation occurs [7]. Biochemical data suggest that during NHEJ pol μ fills gaps at the junction sites [8], [9] and promotes annealing (a few bases) between DNA ends [10]. In agreement with DNA synthesis activity, overexpression of pol μ in cell culture affects the repair of chromosomal ends that require gap filling [11]. However, these repair events are characterized by high levels of DNA synthesis at junction sites that were not reported in vivo [1], and at physiological concentrations of the polymerase, for example for the repair of chromosomal breaks in embryonic stem (ES) cells [12] and in the CHO cell line [13]. Importantly, pol μ is the only tested DNA polymerase able to synthesize in vitro on non-complementary 3′ overhangs (gapped DNA synthesis, also called synthesis on unpaired or non-aligned DNA ends [14]). This property led to the suggestion that pol μ specifically contributes to NHEJ by synthesizing on unpaired DNA ends that cannot be processed by other DNA polymerases. Gapped DNA synthesis was also reported at DSBs on chromosomes [12], although the polymerase responsible for this activity was not identified.
In agreement with the role of pol μ essentially as a end processing enzyme, the repair of complementary or partially complementary ends is not affected by overexpression of pol μ [11]. However, this data contrasts with the DNA annealing activity of pol μ reported in vitro [10] that is expected to affect the repair of complementary ends, although the function of annealing by pol μ has not been shown to directly impact on NHEJ, nor it has been confirmed in a physiological context.
Pol μ seems to act in concert with the other NHEJ components. In particular, it has been suggested that pol μ works together with core NHEJ factors to bridge non-complementary DNA ends and perform a template-dependent gap fill-in reaction [15]. It has also been found that in human crude extracts, gap filling by pol μ is dependent on the presence of Cernunnos [16]. In vivo data also show that pol μ limits the resection (by 6 bp) at Igκ junction sites [1], but in vitro experiments failed to show the same activity, suggesting that end-protection by pol μ may be limited to the chromatin structure of some Ig regions. Thus, in vitro data suggest that the major role of pol μ is to repair some non-complementary DNA ends, essentially by DNA synthesis, and also to promote end annealing. Interestingly, it was reported that pol μ affects the overall efficiency of γ-irradiation induced DSBs in haematopoietic and non-haematopoietic cells [2]. Moreover, we previously showed that pol μ affects the kinetics of radiation-induced DSB repair and reduces the number of residual DSBs long after irradiation [3], suggesting a more global role of pol μ in NHEJ. To clarify the function of pol μ in NHEJ and assess whether pol μ acts other than by DNA synthesis and promoting annealing, we performed a quantitative molecular analysis of DSB repair in wild type and pol μ−/− primary cells using a variety of NHEJ substrates. Our data show that in a cellular context, thereby at physiological concentrations of the repair factors, pol μ intervenes in the repair of all tested DNA ends, including those that did not undergo end processing, suggesting that this DNA polymerase acts as a global player rather than just as a end-processing enzyme in NHEJ.
Section snippets
Cells and culture conditions
Primary wild type and pol μ−/− MEFs, described in Bertocci et al. [1] were cultured in DMEM medium (Dulbecco's modified Eagle's medium, Gibco) supplemented with 10% foetal calf serum (Gibco), 1 mM sodium pyruvate and 50 μg/ml gentamicin at 37 °C with 5% CO2 and 20% O2. Freshly prepared MEFs, originated from different preparations have been amplified twice (dilution 1:3) and then frozen; thawed aliquots were used for experiments. When indicated, either wortmannin (Sigma), or NU7441 (Axon Medchem)
Validation of NHEJ substrates for the analysis of DSB repair.
To examine the role of pol μ in global DSB repair and in a cellular context, we made use of an improved plasmid-based strategy involving clonal DSB repair events in living cells. For this, we generated a variety of NHEJ substrates that were transiently transfected in cells in culture. Distinct from other plasmids that have been widely used to analyse NHEJ in living cells (e.g. [6], [18], [21]), these plasmids do not replicate in mammalian cells, consequently, each retrieved molecule corresponds
Discussion
To dissect the mechanism of action of pol μ our molecular approach differs from others previously described in that NHEJ was performed on a variety of substrates directly in living cells. Importantly, we report that with this strategy all DNA ends were repaired efficiently in a wild type context, similar to chromosomal DSBs [13]. In addition, faithful events were observed at frequencies consistent with chromosomal DSBs [13], and in contrast to the repair by cell-free extracts [22]. This assay
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
We thank B. Bertocci for the generous gift of MEFs and for stimulating discussion. This work was supported by EDF-Radioprotection and Ligue Nationale contre le Cancer. RC was recipient of MESR (Ministère de l’Enseignement Supèrieur et de la Recherche) PhD and Pasteur-Weizmann fellowships.
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