BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis

Background

Breast Cancer Type I Susceptibility Protein (BRCA1) is a tumor suppressor factor whose mutations predispose to the development of cancers. In addition to regulating transcription and the cell cycle, BRCA1 participates in maintaining genome stability by regulating the response to replication stress and DNA double-strand breaks (DSBs) (Mullan et al., 2006; Quinet et al., 2021; San Martin Alonso and Noordermeer, 2021).

In response to DSBs, BRCA1 is recruited to the sites of DNA damage where it regulates different steps of DNA repair during homologous recombination (HR). This 1863 amino acid-long protein has multiple domains that allow its association with distinct complexes that regulate HR either negatively or positively. For example, its association with BRCA1-Associated RING Domain Protein 1 (BARD1) stimulates RAD51-mediated homologous DNA pairing (Zhao et al., 2017)

BRCA1 also plays a crucial role in the cellular response to replication stress by protecting reversed forks ends from nucleolytic attacks orchestrated by nucleases such as MRE11. Interestingly, this protective effect of BRCA1 towards replication forks also requires its interaction with BARD1, which controls RAD51 loading to damaged forks (Daza-Martin et al., 2019).

 

Remarkably, the BRCA1/BARD1 complex displays E3 ubiquitin ligase activity whose function or targets in response to DNA damage are still being investigated. Indeed, research in this area has yielded conflicting results. Some studies have demonstrated that the E3 ubiquitin ligase activity of the complex is required for the repair of double-strand breaks (DSBs) through homologous recombination (HR), while other studies have reported that this activity is dispensable (reviewed in (San Martin Alonso and Noordermeer, 2021; Daniel Salas-Lloret et al., 2023)).

 

In this preprint, the González-Prieto Lab sheds light on the functions and targets of the E3-ligase activity of the BRCA1/BARD1 complex and thus on its mechanism of action in preventing genome instability (Daniel Salas-Lloret et al., 2023). In a previous study, the authors developed an elegant and powerful technique which enables the unambiguous identification of direct ubiquitylation substrates for an E3 of interest through mass spectrometry, named TULIP2 (Targets of Ubiquitin Ligases Identified by Proteomics 2) (Fig. 1A; (Salas-Lloret et al., 2019)). Using this technique, the authors could now reveal that the E3 ubiquitin ligase activity of the BRCA1/BARD1 complex is not required for HR-mediated DSB repair. Instead, they identified that this complex targets PCNA for ubiquitylation to prevent the accumulation of ssDNA gaps during replication and following replication stress.

 

Key findings

1. The E3 ubiquitin ligase activity of the BRCA1/BARD1 complex is not involved in HR-mediated DSBs repair.

To assess whether the E3 ligase activity of the BRCA1/BARD1 complex is involved in HR-mediated DSB repair, the authors used BRCA1 knockout RPE-1 cells supplemented (or not) with the wild type form of BRCA1 or its isoleucine 26 mutated form (I26A). This mutation obliterates the E3-ligase activity of the BRCA1/BARD1 complex without affecting the interaction of these factors. Remarkably, this mutated form of BRCA1 restores the homologous recombination (HR) DSB repair deficiency of BRCA1-KO cells to an equivalent level observed in the same cells complemented with the wild type (Fig. 1B). Thus, the authors demonstrate that the E3-ligase activity of the BRCA1/BARD1 complex is not required during this repair process.

 

2. The BRCA1/BARD1 ubiquitin ligase targets PCNA and protects the genome from the accumulation of ssDNA gaps

In order to figure out how a defect in the E3-ligase activity of this complex predisposes to the development of cancers, the authors decided to identify its targets using their previously developed approach, TULIP2 (Fig. 1A). Following a recent study showing that it is BARD1 – not BRCA1 – that guides the E2 ubiquitin-conjugating enzyme to targets such as histone H2A, the authors integrated BARD1 into the TULIP2 system. As expected, the authors identified histones H2A and macroH2A as substrates, as well as PCNA that appeared to be a primary target of the BRCA1/BARD1 complex. Furthermore, the authors showed that it ubiquitylates PCNA on lysine 164 (K164) under unperturbed conditions, which would facilitate DNA synthesis during replication. In line with the latter hypothesis and data from the literature, the authors showed that BRCA1, the E3 ligase activity of the BRCA1/BARD1 complex and the ubiquitylation of PCNA are required to limit the formation of ssDNA gaps both under unperturbed and replication stress conditions (Fig. 1C).

Finally, the authors could reveal that the E3 ligase activity of the BRCA1/BARD1 complex is required for its phosphorylation (phospho-BRCA1 S114) which has been described to regulate its isomerization that triggers its interaction with RAD51 and regulates replication forks protection in response to replication stress.

3. Conclusion

By showing that the E3 ligase activity of the BRCA1/BARD1 complex is not involved in DSB repair during HR but is required for genome stability during replication and in response to replication stress, the authors shed new light on the mechanisms by which BRCA1 and BARD1 maintain genome stability and prevent cancer occurrence.

Figure 1. The BRCA1/BARD1 complex targets PCNA for ubiquitylation to prevent ssDNA gaps accumulation during replication (with panels from (Daniel Salas-Lloret et al., 2023))

A. Illustration of the TULIP2 system and the methodology for unambiguously identifying the targets of an E3-ligase of interest (adapted from (Salas-Lloret et al., 2019; Daniel Salas-Lloret et al., 2023)). The DNA coding for the E3-ligase of interest is cloned into a lentiviral vector followed by a sequence coding for ubiquitin and flanked on both sides by a 10HIS tag. These two 10HIS tags allow nickel-based purification to be carried out under denaturing conditions and thus eliminate non-specific and non-covalent protein-protein interactions. Genome integration of the TULIP2 system after viral infection and doxycycline-induced expression allows the expression level of the recombinant E3 ligase to be controlled at a near-endogenous level.

B. Representative micrographs of immunofluorescence to monitor RAD51 foci formation following ionizing radiation (10Gy) in Parental and BRCA1-KO cells complemented (or not) with either BRCA1-WT-GFP or BRCA1-I26A-GFP.

C. Representative micrographs of immunofluorescence to monitor ssDNA levels following (or not) DNA replication stress induced by hydroxyurea (HU) in Parental and BRCA1-KO cells complemented (or not) with either BRCA1-WT-GFP or BRCA1-I26A-GFP.

References

Daniel Salas-Lloret, Nestor Garcia-Rodriguez, Lisanne Giebel, Arnoud de Ru, Peter A. van Veelen, Pablo Huertas, et al. (2023). BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis. BioRxiv.

Daza-Martin, M., Starowicz, K., Jamshad, M., Tye, S., Ronson, G. E., MacKay, H. L., et al. (2019). Isomerization of BRCA1–BARD1 promotes replication fork protection. Nature 571, 521–527. doi: 10.1038/s41586-019-1363-4.

Mullan, P. B., Quinn, J. E., and Harkin, D. P. (2006). The role of BRCA1 in transcriptional regulation and cell cycle control. Oncogene 25, 5854–5863. doi: 10.1038/sj.onc.1209872.

Quinet, A., Tirman, S., Cybulla, E., Meroni, A., and Vindigni, A. (2021). To skip or not to skip: choosing repriming to tolerate DNA damage. Mol Cell 81, 649–658. doi: 10.1016/j.molcel.2021.01.012.

Salas-Lloret, D., Agabitini, G., and González-Prieto, R. (2019). TULIP2: An Improved Method for the Identification of Ubiquitin E3-Specific Targets. Front Chem 7. doi: 10.3389/fchem.2019.00802.

San Martin Alonso, M., and Noordermeer, S. M. (2021). Untangling the crosstalk between BRCA1 and R-loops during DNA repair. Nucleic Acids Res 49, 4848–4863. doi: 10.1093/nar/gkab178.

Zhao, W., Steinfeld, J. B., Liang, F., Chen, X., Maranon, D. G., Jian Ma, C., et al. (2017). BRCA1–BARD1 promotes RAD51-mediated homologous DNA pairing. Nature 550, 360–365. doi: 10.1038/nature24060.

 

 

What I liked about this preprint

The data from the González-Prieto Lab are of great interest to the research fields of DNA damage repair and replication. Thanks to their elegant and powerful method, the authors have obtained results that allow to conclude that the BRCA1/BARD1 complex participates in genome stability, at least via its E3-ligase activity during replication and independently of its ligase activity during HR. In particular, these data allow the consideration of new therapeutic options depending on the mutations carried in the genes coding for BRCA1 and BARD1.

 

Questions to the authors

Q1: Several mutually exclusive mechanisms have been described for maintaining replication fork stability and processivity under replicative stress, including fork reversal, repriming DNA synthesis and Trans-lesion synthesis (TLS). Could you share your thoughts on how BRCA1/BARD1-mediated PCNA ubiquitylation may regulate TLS? Finally, do you think that the E3 ligase activity of the BRCA1/BARD1 complex can be seen as the cornerstone that regulates the choice between fork reversal, repriming DNA synthesis and TLS?

Q2: Concerning the analysis of mass spectrometry results. Is it possible to identify the types of ubiquitin chains (Ub-K63, Ub-K48,…) that form on the targets identified by mass spectrometry? Conversely, would you recommend the use of ubiquitin mutants that allow the assembly of only one type of chain if one wishes to answer this question while using the TULIP2 system?

Tags: brca1/bard1, e3 ubiquitin ligase, replication

doi: https://doi.org/10.1242/prelights.34041

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