RNA-splicing factor SART3 regulates translesion DNA synthesis

Abstract

Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance that uses specialized DNA polymerases to replicate damaged DNA. DNA polymerase η (Polη) is well known to facilitate TLS across ultraviolet (UV) irradiation and mutations in POLH are implicated in skin carcinogenesis. However, the basis for recruitment of Polη to stalled replication forks is not completely understood. In this study, we used an affinity purification approach to isolate a Polη-containing complex and have identified SART3, a pre-mRNA splicing factor, as a critical regulator to modulate the recruitment of Polη and its partner RAD18 after UV exposure. We show that SART3 interacts with Polη and RAD18 via its C-terminus. Moreover, SART3 can form homodimers to promote the Polη/RAD18 interaction and PCNA monoubiquitination, a key event in TLS. Depletion of SART3 also impairs UV-induced single-stranded DNA (ssDNA) generation and RPA focus formation, resulting in an impaired Polη recruitment and a higher mutation frequency and hypersensitivity after UV treatment. Notably, we found that several SART3 missense mutations in cancer samples lessen its stimulatory effect on PCNA monoubiquitination. Collectively, our findings establish SART3 as a novel Polη/RAD18 association regulator that protects cells from UV-induced DNA damage, which functions in a RNA binding-independent fashion.

Mechanisms of DNA Damage Tolerance: Post-Translational Regulation of PCNA

“…Through affinity purification and mass spectrometry, SIVA1 (SIVA apoptosis-inducing factor 1), SART3 (squamous cell carcinoma antigen recognized by T Cells 3), and MAGE-A4 (melanoma associated antigen-A4) were identified as critical regulators of PCNA mono-ubiquitination in response to UV-induced DNA damage [109,110,111]. SIVA1 plays an important role in the extrinsic and intrinsic apoptotic pathways induced by the CD27 (cluster of differentiation 27) receptor, whereas SART3, a pre-mRNA splicing factor, functions in the recycling of the splicing machinery.…”

“…SIVA1 plays an important role in the extrinsic and intrinsic apoptotic pathways induced by the CD27 (cluster of differentiation 27) receptor, whereas SART3, a pre-mRNA splicing factor, functions in the recycling of the splicing machinery. Outside their known roles in apoptosis and mRNA splicing, SIVA1 and SART3 function as adaptors for Rad18 to bind to PCNA, and promote efficient PCNA ubiquitination in response to UV radiation [109,110]. Other factors, such as MAGE-A4 do not function as a bridge between Rad18 and PCNA, but instead directly bind to Rad18 to stabilize it.…”

Increased versatility despite reduced molecular complexity: evolution, structure and function of metazoan splicing factor PRPF39

“…HAT-repeat proteins are mostly involved in protein-protein and protein-RNA interactions and act as scaffolding proteins. They participate in various RNA processing pathways, prominent examples being SART3, which is involved in splicing and translesion DNA synthesis (11) and RNA14, which is important for mRNA 3′-end maturation (12). Furthermore, a cryo-EM structure of a yeast pre-catalytic pre-B complex (13) revealed that the contact between the U1 and U2 snRNP is largely mediated by the yPrp42/yPrp39 heterodimer.…”

Lsm12 mediates Pol·q deubiquitination to help Saccharomyces cerevisiae resist oxidative stress

“…Similar to TS, HR also belongs to the error-free branch of the DNA damage tolerance pathway (13). In contrast, TLS is referred to as the error-prone branch of DNA damage tolerance (14), and is a conserved mechanism from bacteria to mammals that recruits various specialized DNA polymerases to the stalled replication forks (15–17). These specialized polymerases mostly belong to the Y family, consisting of Polη and Rev1 in yeasts, encoded by RAD30 and REV1 , respectively (18).…”