Abstract:

The DNA damage checkpoint is a signalling cascade that is crucial for the maintenance of genome stability after genotoxic stress; without it, cells cannot complete genome duplication. The lethal consequences of not having the checkpoint can be suppressed in human cells by preventing the firing of excess origins of DNA replication. We show that this is because the activation of excess origins produces large numbers of Okazaki fragments, which sequester Replication Factor C (RFC) that loads the polymerase processivity factor known as PCNA. Sequestering RFC in the absence of the checkpoint then blocks subsequent fork restart and leaves unprotected ends of nascent DNA that are vulnerable to nucleolytic attack. Through a genome-wide siRNA screen, we identified the ‘Helicase-Like Transcription Factor’ (HLTF) as the main enzyme that attacks unprotected 3’ ends in absence of the checkpoint. Loss of HLTF suppresses aberrant restart, accumulation of ssDNA, DNA damage, and cell lethality in checkpoint-deficient cells. To explore the mechanism in detail, we reconstituted fork stalling and restart in vitro with purified proteins from budding yeast. We show that leading strand DNA synthesis stops quickly under such conditions, whilst continued DNA unwinding allows the initiation of many new Okazaki fragments that sequester the processive DNA synthesis machinery (PCNA, RFC, Polδ, and Polε), exposing nascent DNA to enzymatic attack and blocking the restart of processive leading strand synthesis. This work has implications for how origin firing and replication forks are regulated during unperturbed S phase, the process of fork reversal at stalled forks, and how resistance to anti-checkpoint therapies might arise in human cancer patients.

Biography:

Agos completed her doctoral studies at the Leloir Institute in Buenos Aires, Argentina. During her PhD, she investigated the post-translational regulation of the transcription factor HIF-1 (Hypoxia Inducible Factor-1), using Drosophila melanogaster as a model organism. Following her PhD, Agos undertook a short postdoctoral position in Vanesa Gottifredi’s lab, where she researched the role of the Translesion Synthesis DNA Polymerase Iota in human cells. In 2017, she relocated to London to join the Chromosome Replication Lab at the Francis Crick Institute, led by John Diffley. Using two completely different approaches — biochemistry with purified yeast proteins (collaboration with Berta Canal in the Diffley lab), and cell biology and genetic screens using human tissue culture cells, Agos has addressed a question that has puzzled the genome stability field for over two decades: how does the DNA damage checkpoint prevent the irreversible collapse of stalled DNA replication forks?

Agos also contributed to a multi-centre translational collaboration during the COVID lockdowns in 2020, to screen for inhibitors of the enzymes encoded by the SARS-CoV-2 virus.

Publications:

A mitotic stopwatch determines cell fate. Bertolin AP, Gottifredi V. Science. 2024 Mar 29;383(6690):1414-1415. doi: 10.1126/science.ado5703. Epub 2024 Mar 28.

Polymerase iota (Pol ι) prevents PrimPol-mediated nascent DNA synthesis and chromosome instability. Mansilla SF, Bertolin AP, Venerus Arbilla S, Castaño BA, Jahjah T, Singh JK, Siri SO, Castro MV, de la Vega MB, Quinet A, Wiesmüller L, Gottifredi V. Sci Adv. 2023 Apr 14;9(15):eade7997. doi: 10.1126/sciadv.ade7997. Epub 2023 Apr 14.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase. Bertolin AP, Weissmann F, Zeng J, Posse V, Milligan JC, Canal B, Ulferts R, Wu M, Drury LS, Howell M, Beale R, Diffley JFX. Biochem J. 2021 Jul 16;478(13):2425-2443. doi: 10.1042/BCJ20210200.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase. Zeng J, Weissmann F, Bertolin AP, Posse V, Canal B, Ulferts R, Wu M, Harvey R, Hussain S, Milligan JC, Roustan C, Borg A, McCoy L, Drury LS, Kjaer S, McCauley J, Howell M, Beale R, Diffley JFX. Biochem J. 2021 Jul 16;478(13):2405-2423. doi: 10.1042/BCJ20210201.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease. Canal B, Fujisawa R, McClure AW, Deegan TD, Wu M, Ulferts R, Weissmann F, Drury LS, Bertolin AP, Zeng J, Beale R, Howell M, Labib K, Diffley JFX. Biochem J. 2021 Jul 16;478(13):2465-2479. doi: 10.1042/BCJ20210199.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease. Canal B, McClure AW, Curran JF, Wu M, Ulferts R, Weissmann F, Zeng J, Bertolin AP, Milligan JC, Basu S, Drury LS, Deegan TD, Fujisawa R, Roberts EL, Basier C, Labib K, Beale R, Howell M, Diffley JFX. Biochem J. 2021 Jul 16;478(13):2445-2464. doi: 10.1042/BCJ20210198.

Under-Replicated DNA: The Byproduct of Large Genomes? Bertolin AP, Hoffmann JS, Gottifredi V. Cancers (Basel). 2020 Sep 25;12(10):2764. doi: 10.3390/cancers12102764.

Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster. Beckwith EJ, Hernando CE, Polcowñuk S, Bertolin AP, Mancini E, Ceriani MF, Yanovsky MJ. Genetics, 2017 Oct;207(2):593-607. doi: 10.1534/genetics.117.300139. Epub 2017 Aug 11.

Musashi mediates translational repression of the Drosophila hypoxia inducible factor. Bertolin AP, Katz MJ, Yano M, Pozzi B, Acevedo JM, Blanco-Obregón D, Gándara L, Sorianello E, Kanda H, Okano H, Srebrow A, Wappner P. Nucleic Acids Res. 2016 Sep 19;44(16):7555-67. doi: 10.1093/nar/gkw372. Epub 2016 May 3.

miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga. De Lella Ezcurra AL, Bertolin AP, Kim K, Katz MJ, Gándara L, Misra T, Luschnig S, Perrimon N, Melani M, Wappner P. PLoS Genet. 2016 May 25;12(5):e1006073. doi: 10.1371/journal.pgen.1006073.

Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells. Mansilla SF, Bertolin AP, Bergoglio V, Pillaire MJ, González Besteiro MA, Luzzani C, Miriuka SG, Cazaux C, Hoffmann JS, Gottifredi V. Elife. 2016 Oct 14;5:e18020. doi: 10.7554/eLife.18020.

The identification of translesion DNA synthesis regulators: Inhibitors in the spotlight. Bertolin AP, Mansilla SF, Gottifredi V. DNA Repair (Amst). 2015 Aug;32:158-164. doi: 10.1016/j.dnarep.2015.04.027. Epub 2015 May 12.

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation. Vallerga MB, Mansilla SF, Federico MB, Bertolin AP, Gottifredi V. Proc Natl Acad Sci U S A. 2015 Dec 1;112(48):E6624-33. doi: 10.1073/pnas.1508543112. Epub 2015 Nov 16.

Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia. Dekanty A, Romero NM, Bertolin AP, Thomas MG, Leishman CC, Perez-Perri JI, Boccaccio GL, Wappner P. PLoS Genet. 2010 Jun 24;6(6):e1000994. doi: 10.1371/journal.pgen.1000994.