Development of an in vitro assay for detection of translesion synthesis in human mitochondrial extracts and identification of the polymerases involved

• Grant number: 22/16432-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2023
• End date: March 31, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Nadja Cristhina de Souza Pinto
• Grantee: Beatriz Kopel
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Most organisms store their genetic information in DNA, which is constantly exposed to endogenous and exogenous physical and chemical agents that damage it. DNA lesions can block replication or transcription, leading to dysfunction and cell death, or can be potentially mutagenic. In addition to DNA repair pathways, cells have DNA damage tolerance mechanisms (DDT) that allow DNA replication even in the presence of lesions. Replication blockage by DNA damage can be alleviated through two distinct DDT mechanisms: template switching (TS) and translesion synthesis (TLS). In the TLS mechanism, the replicative DNA polymerase is temporarily replaced by one or two specialized DNA polymerases capable of synthesizing past the lesion, but since these polymerases lack proof-reading activity, this process is potentially mutagenic. The mitochondrial DNA (mtDNA) is a primary target of reactive oxygen species generated during oxidative phosphorylation, in addition to being preferentially damaged in cells exposed to alkylating and oxidizing agents, such that maintaining mtDNA integrity is essential for cellular homeostasis. Some repair pathways such as base excision, mismatch and double-strand break repair have already been characterized in mitochondria, however, there is still much to be elucidated. DDT requires the activity of DNA polymerases, either for TS or TLS movements. From the 17 known human DNA polymerases, in addition to the replicative DNA polymerase gamma, there is evidence of mitochondrial localization of primase-polymerase (PrimPol) and DNA polymerases beta, zeta, eta and theta. The latter are TLS polymerases in the nucleus, but their TLS activity in mitochondria have not been yet demonstrated. The few available evidences of TLS in mitochondria are attributed only to DNA polymerase gamma activity. However, the participation of any other DNA polymerase in TLS on mtDNA has not yet been demonstrated. Thus, the goal of this project is to investigate the participation of DNA polymerases with supposed mitochondrial localization in translesion synthesis in mtDNA.