investigation-protein-recruitment-to-dna-lesions-using-405-nm-laser

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Université de Sherbrooke

Cancer cells overcome replicative senescence by exploiting mechanisms of telomere elongation, a process often accomplished by reactivation of the enzyme telomerase. However, a subset of cancer cells lack telomerase activity and rely on the alternative lengthening of telomeres (ALT) pathway, a recombination-based mechanism of telomere elongation. Although the mechanisms regulating ALT are not fully defined, chronic replication stress at telomeres might prime these fragile regions for recombination. Here, we demonstrate that the replication stress response protein SMARCAL1 is a critical regulator of ALT activity. SMARCAL1 associates with ALT telomeres to resolve replication stress and ensure telomere stability. In the absence of SMARCAL1, persistently stalled replication forks at ALT telomeres deteriorate into DNA double-strand breaks promoting the formation of chromosome fusions. Our studies not only define a role for SMARCAL1 in ALT telomere maintenance, but also demonstrate that resolution of replication stress is a crucial step in the ALT mechanism.

SMARCAL1 Resolves Replication Stress at ALT Telomeres.

RPA-coated single-stranded DNA (RPA-ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA-ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR.

Article	Year
SMARCAL1 Resolves Replication Stress at ALT Telomeres. Cell reports\| PubMed ID: 26832416	2016
A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination. Nucleic acids research\| PubMed ID: 28666352	2017

Alexandre Marechal

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