Investigation of the role of alkyladenine DNA glycosylase (AAG) in the repair of alkylated bases in mice mitochondrial DNA

Author(s)

Rebeca Bueno Alves

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2021-09-24

Examining board members

Nadja Cristhina de Souza Pinto; Roger Chammas; Nicolas Carlos Hoch; Lisiane Borges Meira

• Advisor: Nadja Cristhina de Souza Pinto

Abstract

Living organisms are constantly exposed to agents that can damage their DNA. DNA repair mechanisms contribute to maintain genomic stability. The mitochondrial DNA (mtDNA) is also subject to damage and may accumulate more lesions than nuclear DNA, likely due to its location, close to the reactive oxygen species-generating electron transport chain. In mitochondria, the main DNA repair pathway is the base excision repair (BER), which repairs single strand breaks and base modifications such as oxidations and alkylations. BER is initiated by the recognition of modified bases by a DNA glycosylase. Alkylated bases, like 3- methyladenine, a cytotoxic DNA lesion, are recognized and repaired by the alkyladenine DNA glycosylase (AAG). Changes in DNA repair are implicated in human diseases and impact therapeutic efficiency. For instance, AAG activity can modulate the efficiency of various chemotherapeutic agents. These studies are usually conducted in murine models. AAG has been localized to mitochondria and nuclei in human cells, but mitochondrial localization in murine mitochondria after alkylation damage has not yet been detected. Thus, in the present work we sought to investigate the role of AAG in mtDNA repair in mouse cells (C2C12) treated with the alkylating agent methylmethanesulfonate (MMS). MMS-induced lesion formation and repair were measured using long-range PCR (XL-PCR), which is based on the principle that lesions that block the DNA polymerase progression will affect the amplification of a long DNA fragment. The results obtained showed that MMS induces lesion formation in mouse mtDNA, which are completely removed after 24 hours, as observed in the nucleus. There is no mtDNA depletion, as measured by mtDNA copy number, suggesting that MMS-induced lesions are being removed by DNA repair, rather than by degradation of the affected DNA. To ascertain whether AAG is the enzyme responsible for removing these lesions, AAG intracellular localization was evaluated by immunofluorescence. Results showing cytosolic AAG localization suggest a possible mitochondrial localization of AAG in these organisms. (AU)

• FAPESP's process: 18/23744-9 - Investigation of the role of Alkyladenine DNA glycosylase (AAG) in the repair of alkylated bases in the mitochondrial DNA of mice
• Grantee: Rebeca Bueno Alves
• Support Opportunities: Scholarships in Brazil - Master