Investigating The Role Of SIVA1 In Translesion DNA Synthesis

Our genome is continuously challenged by endogenous and exogenous agents, such as misincorporated nucleotides raised in replication, free radicals generated from metabolism, ultraviolet light and ion radiation. These factors will lead to many different types of lesions which can block both replication and transcription. Untimely or inaccurate repair of DNA damage can result in gene mutations, chromosome aberrations, cell senescence or apoptosis. To counteract DNA damage, cells have evolved a variety of highly developed surveillance and repair pathways to maintain genome integrity.Translesion DNA synthesis (TLS) is a universal DNA damage tolerance (DDT) mechanism conserved throughout evolution. It relies on a class of specialized DNA polymerases termed as TLS polymerases. These TLS polymerases possess a spacious active site capable of accommodating damaged templates that would block the high-fidelity replicative DNA polymerases. Most of the TLS polymerases belong to the Y family, which includes Polη, Poll, Polk, and REV1. Each of the TLS polymerases has different specificities for different types of DNA damage. For example, human Polη plays a key role in the accurate replication of cis-syn thymine-thymine (T-T) dimer, a major photoproduct induced by UV irradiation. Inactivation of Polη in humans causes the variant form of the skin cancer-prone syndrome xeroderma pigmentosum (XP-V), characterized by hypersensitivity to UV exposure and an increased incidence of skin cancers.A key event in the regulation of TLS is the monoubiquitination of proliferating cell nuclear antigen (PCNA) at the conserved lysine 164 by the E3 ubiquitin ligase RAD 18 and the E2 ubiquitin conjuncting enzyme RAD6. PCNA monoubiquitination serves to recruit Y family TLS polymerases to trigger lesion bypass. Y family TLS polymerases efficiently compete with replicative polymerases for interaction with monoubiquitinated PCNA through their ubiquitin binding UBM or UBZ domain in addition to their PIP box (PCNA-interacting peptide box).However, the mechanism by which the RAD6-RAD18 complex is targeted to PCNA has remained elusive. In this study, we used an affinity purification approach to isolate the PCNA-containing complex and have identified SIVA1 as a critical regulator of PCNA monoubiquitination. We show that SIVA1 constitutively interacts with PCNA via a highly conserved PIP box. Knockdown of SIVA1 compromised RAD 18-dependent PCNA monoubiquitination and Polr1 focus formation, leading to elevated ultraviolet sensitivity and mutation. Furthermore, we demonstrate that SIVA1 interacts with RAD 18 and serves as a molecular bridge between RAD 18 and PCNA, thus targeting the E3 ligase activity of RAD 18 onto PCNA. Collectively, our results provide evidence that the RAD 18 E3 ligase requires an accessory protein for binding to its substrate PCNA. These findings will provide new insights into the molecular mechanisms of TLS pathway, which is critically important for the maintenance of genome integrity and tumor suppression.