Analysis of the role of the XPG endonuclease in nuclear transcription and mitochondrial genome integrity

Author(s)

Davi Mendes

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2024-02-07

Examining board members

Carlos Frederico Martins Menck; Mário Henrique de Barros; José Renato Rosa Cussiol; Nicolas Carlos Hoch

• Advisor: Carlos Frederico Martins Menck

Abstract

DNA repair mechanisms are vital for maintaining genomic integrity and preventing the accumulation of DNA lesions that can lead to a wide range of diseases, including cancer, aging-related disorders, and neurodegenerative conditions. Nucleotide excision repair (NER) is one of the key DNA repair systems responsible for recognizing and repairing a variety of DNA lesions caused by exposure to UV radiation, chemical agents, or oxidative stress. Deficiencies in NER proteins can result in disorders such as Xeroderma Pigmentosum (XP), characterized by extreme sensitivity to UV light and an increased risk of skin cancer, and Cockayne Syndrome (CS), associated with severe neurodevelopmental and premature aging features. To investigate the roles of specific NER proteins, such as XPG, in dealing with redox stress, cell lines were generated by transducing XPG WT and XPG RJ2 (XP) alleles to a cell line XPG deficient, from a XP/CS patient. First, they were evaluated after UV exposure, followed by assessment at basal levels and after redox stress. Various cellular parameters were evaluated, including cell viability, cell cycle and &#947H2AX levels. Fluorescent probes, antibodies targeting mitochondrial proteins, and immunofluorescence analysis were utilized to examine radical oxygen species (ROS) production, mitochondrial features, and redox stress sensitivity. Host cell reactivation (HCR) assay was employed to evaluate NER capacity, while alkaline comet assays, combined with Fpg enzyme treatment, allowed the assessment of DNA breaks, oxidized bases, and repair kinetics. The results of this study provided important insights into the impact of XPG alleles on redox stress, mitochondrial parameters, and cellular metabolism. It was observed that the XPG WT allele increased survival and reduced cellular oxidative stress without increasing redox defenses, while ROS production did not result from mitochondrial dysfunction. Interestingly, XPG WT cells exhibited improved mitochondrial and non-mitochondrial respiration, suggesting a potential metabolic benefit due to complementation. In contrast, XPG RJ2 increased cell death, demonstrated similar profile of XPG/CS cells, except by a partial metabolic improvement with increased spare capacity and maximal respiration. Furthermore, XPG/CS cells exhibited increased pyruvate dehydrogenase (PyD) expression, likely in an adaptive response to survive redox stress. The exact trigger for this response remains to be elucidated, and further investigations are needed to uncover the underlying mechanisms driving these observations. These findings shed light on the intricate relationships between DNA repair systems, cellular responses to redox stress, and the maintenance of genomic integrity. Understanding the roles of specific NER proteins in these processes has important implications for help elucidating the mechanisms of DNA repair and their contributions to neurodegeneration and aging. (AU)

• FAPESP's process: 17/18781-0 - The role of XPG endonuclease in nuclear RNA transcription and in the maintenance of mitochondrial genome
• Grantee: Davi Mendes
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)