During DNA replication, DNA lesions can block the progression of replication forks. Replication fork stalling in turn leads to an accumulation of extensive single-stranded DNA (ssDNA) regions, which are bound and stabilized by the RPA trimer (Figure 2) (Ciccia and Elledge, Mol Cell, 2010). RPA can then facilitate the activation of the ATR kinase by the RAD9-HUS1-RAD1 (9-1-1) complex together with TOPBP1. Induction of the DDR after replication stress leads to the protection of replication forks, repair of DNA lesions and restart of DNA synthesis.
Figure 2. Simplified representation of the DNA damage response at stalled replication forks
SMARCAL1 AND ZRANB3
The ssDNA binding complex RPA is a critical regulator of replication fork stability and restart. We have previously reported the identification of SMARCAL1 as a novel RPA interactor that is recruited to replication forks blocked by DNA lesions, where it promotes the restart of DNA synthesis (Ciccia et al., Genes Dev, 2009). In these studies, replication fork restart was monitored on single DNA fibers from cells incubated with the nucleotide analog IdU, treated with the DNA polymerase inhibitor aphidicolin and subsequently supplemented with the nucleotide analog CldU following aphidicolin removal (Figure 3).
SMARCAL1 is a DNA translocase mutated in Schimke immuno-osseous dysplasia (SIOD), a rare recessive childhood disorder characterized by growth defects, renal dysfunction, T-cell immunodeficiency, microcephaly and predisposition to malignancy (Boerkoel et al., Nat Genetics, 2002). In our work, we showed that the defective proliferation exibited by SIOD patient cells is due to spontaneous replication fork damage. These studies elucidated how RPA facilitates the restart of stalled replication forks and provided new insights into the pathogenesis of the SIOD disorder.
Figure 3. SMARCAL1 promotes the restart of stalled replication forks in mammalian cells
Completion of DNA replication after DNA damage is regulated by the DNA polymerase clamp PCNA, which is subjected to mono- or poly-ubiquitination to promote error-prone or error-free bypass of DNA lesions, respectively (Ciccia and Elledge, Mol Cell, 2010). We have previously described the identification of ZRANB3, a DNA translocase related to SMARCAL1, as the first mammalian protein that associates with poly-ubiquitinated PCNA to restart replication forks blocked by DNA lesions (Ciccia et al, Mol Cell, 2012). In these studies, we have shown that both ZRANB3 and SMARCAL1 remodel stalled replication forks to favor their restart after DNA damage (Figure 4). Our work provided novel insights into the role of ZRANB3 and SMARCAL1 in preserving replication fork integrity.
Figure 4. SMARCAL1 and ZRANB3 promote fork regression and dissociate recombination intermediates at stalled replication forks
Characterization of the molecular mechanisms that maintain replication fork stability
Maintenance of replication fork stability relies on the complex and still poorly understood interplay between a multitude of DNA repair factors (Ciccia and Elledge, Mol Cell, 2010). To gain a complete understanding of the network that preserves replication fork stability, we are defining the proteomic and genetic interactions exhbited by SMARCAL1, ZRANB3 and other key factors that protect replication forks in response to replication stress. These studies will provide a comprehensive view of the machineries that protect replication forks and will identify new factors that maintain replication fork stability.
Definition of the mechanisms that cause replication fork instability in cancer
BRCA1 and BRCA2 are the most frequently mutated tumor suppressor genes in hereditary breast and ovarian cancer. Recent studies have uncovered a novel role for BRCA1 and BRCA2 in preventing replication fork degradation upon replication stress (Schlacher et al, Cell, 2011; Schlacher et al, Cancer Cell, 2012). We are currently investigating how DNA repair factors interplay with BRCA1 and BRCA2 at stalled replication forks. Our initial studies led to the discovery that SMARCAL1, ZRANB3 and their related factor HLTF promote MRE11-dependent nascent DNA degradation and genomic instability in BRCA1- and BRCA2-deficient cells (Taglialatela, Alvarez et al, Mol Cell, 2017; Figure 5). These studies provide novel insights into the mechanisms by which BRCA1 and BRCA2 maintain genome integrity and prevent the development of breast and ovarian cancer.
Figure 5. Fork reversal induced by SMARCAL1, ZRANB3 or HLTF causes MRE11-dependent fork degradation in BRCA1- and BRCA2-deficient cells
Definition of the underlying causes of human genetic disorders that exhibit replication fork instability
Mutations in genes that preserve replication fork stability cause genetic disorders affecting human development and predisposing to premature aging and cancer, including Bloom and Werner syndromes, Fanconi anemia, Seckel syndrome, Nijmegen breakage syndrome and Schimke immuno-osseous dysplasia (SIOD) (Table 2) (Ciccia and Elledge, Mol Cell, 2010). SMARCAL1 is mutated in approximately half of the cases of SIOD (Clewing et al, Hum Mutat, 2007). We are currently defining the genetic bases for the remaining half of SIOD incidences. Furthermore, we are investigating how SMARCAL1 deficiency causes growth abnormalities and microcephaly using SMARCAL1 knockout mouse models and human cerebral organoids.