DNA can be damaged by numerous endogenous and exogenous factors. To safeguard the genome against these insults, cells have evolved DNA damage checkpoints that sense the presence of damaged DNA, block cell cycle progression and ensure that DNA is fully repaired before resuming the cell cycle. However, in many cases, unrepaired lesions remain in the DNA when cells enter S-phase. In this scenario, cells employ DNA damage tolerance mechanisms to complete genome replication and prevent fork breakage (Figure 1). Importantly, these pathways are not restricted to the site of stalling but can also function behind the fork at single-stranded(ss)DNA gaps originated by re-priming of DNA synthesis downstream of lesions.

While it is well known that ssDNA is the signal that triggers the checkpoint response, it is less clear how and where ssDNA actually arises. Generally, it is assumed to accumulate at stalled replication forks by an uncoupling between replicative helicase and polymerase movement. However, recent evidence found in budding yeast support a model where, in response to polymerase-blocking lesions, replication forks do not stall but recover efficiently by re-priming downstream of the lesions, thus leaving ssDNA gaps behind the fork. Nucleolytic processing of such gaps is then required for efficient checkpoint activation. It is currently unknown whether this model also applies to vertebrate cells, and many questions remain regarding the cellular response to damaged DNA during replication. Where is the checkpoint signaling initiated in human cells, at the fork or behind the fork? What are the molecular mechanisms involved? Do nucleases play a role?

To address these and other questions, we will make use of a unique set of multidisciplinary approaches with the final goal to shed light on the molecular mechanisms of checkpoint activation and damage tolerance during replication of damaged DNA in human cells (Figure 2). An appropriate response to DNA replication stress acts as critical barrier to carcinogenesis. Thus, understanding the pertinent signaling pathways that deal with replicative stress is highly relevant to cell survival and human disease. Interestingly, recent evidence has identified ssDNA gaps as a cancer vulnerability. Thus, by manipulating how gaps are processed, we also hope to open new avenues in the treatment of cancer.

Projects as PI:

• Sensing and dealing with DNA damage during replication as a critical barrier for carcinogenesis. EMERGIA21-00057. Junta de Andalucía.
• Mechanistic analysis of DNA damage signaling and bypass upon replication of damaged DNA template in human cells. DNAcheck-794054. H2020-MSCA-IF-2017. European Commission.

If you are interested in joining our research project as master or PhD student, please contact us at: nestor.garcia@cabimer.es

Articles:

1. Salas-Lloret D*, García-Rodriguez N*, Soto-Hidalgo E, González-Vinceiro L, Espejo-Serrano C, Giebel S, Mateos-Martín ML, de Ru AH, van Veelen P, Huertas P , Vertegaal A, González-Prieto R. (2024). BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis. Nature Communications. (*equal contribution).

2. García-Rodríguez N*, Domínguez-García I, Domínguez-Pérez MdC, Huertas P*. (2024). EXO1 and DNA2-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells. Nucleic Acids Research. (*corresponding authors).

3. Nicastro N, Haillard H, Zarzuela L, Peli-Gulli MP, Fernández-García E, Tomé M, , García-Rodríguez N, Durán RV, De Virgilio C, Wellinger R. (2022). Manganese is a physiologically relevant TORC1 activator in yeast and mammals. eLife.

4. Cano-Linares MI, Yañez-Vilches, A, , García-Rodríguez N, González-Prieto, R, San- Segundo, P, Ulrich, HD, Prado, F (2021). Non-Recombinogenic Roles for Rad52 in Translesion Synthesis during DNA Damage Tolerance. EMBO Reports.

5. Wong RP, García-Rodríguez N, Zilio N, Hanulová M, Ulrich HD (2020). Processing of DNA polymerase-blocking lesions during genome replication is spatially and temporally segregated from replication forks. Molecular Cell.

6. García-Rodríguez N*, Ulrich HD* (2019). A dual system to manipulate protein levels for DNA replication- and cell cycle-related studies. Methods in Enzymology. (*corresponding authors).

7. García-Rodríguez N*, Wong RP, Ulrich HD* (2018). The helicase Pif1 functions in the template switching pathway of DNA damage bypass. Nucleic Acids Research. (*corresponding authors).

8. García-Rodríguez N, Morawska M, Wong RP, Daigaku Y, Ulrich HD (2018). Spatial separation between replisome- and template-induced replication stress signaling. EMBO Journal.

9. García-Rodríguez N*, Wong RP*, Ulrich HD (2016). Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress. Frontiers in Genetics (*equal contribution).

10. Stuckey R*, García-Rodríguez N*, Aguilera A, Wellinger RE (2015). Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system. PNAS. (*equal contribution).

11. García-Rodríguez N, Manzano-López J, Muñoz-Bravo M, Fernández-García E, Muñiz M, Wellinger RE (2015). Manganese redistribution by calcium-stimulated vesicle trafficking bypasses the need for P-type ATPase function. Journal of Biolical Chemistry.

12. Voisset C*, García-Rodríguez N*, Birkmire A, Blondel M*, Wellinger RE*. (2014). Using yeast to model calcium-related diseases: example of the Hailey-Hailey disease. Biochim Biophys Acta. (*equal contribution).

13. García-Rodríguez N, Díaz de la Loza MC, Andreson B, Monje-Casas F, Rothstein R, Wellinger RE. (2012). Impaired manganese metabolism causes mitotic misregulation. Journal of Biolical Chemistry.

A complete list of publications can be obtained at:

https://orcid.org/0000-0002-4049-1604