Characterisation of the role of the ATR kinase homolog in L. major response to replication stress

Author(s)

Gabriel Lamak Almeida da Silva

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-09-14

Examining board members

Luiz Ricardo Orsini Tosi; Leticia Fröhlich Archangelo; Júlia Pinheiro Chagas da Cunha; José Renato Rosa Cussiol

• Advisor: Luiz Ricardo Orsini Tosi

Abstract

Eukaryotic cells have evolved mechanisms to maintain and replicate their genomes, whose integrity and transmission are constantly challenged by DNA damage and replication impediments. The protein kinase Ataxia-Telangiectasia and Rad3-related (ATR), a member of the phosphatidylinositol 3-kinase-like family, ensures genome maintenance and stability, considered as a master regulator of the eukaryotic response to DNA injuries. In this PhD thesis, I investigate the conservation and functional relevance of the ATR homolog in the DNA metabolism of Leishmania major, a protozoan parasite with a remarkably plastic genome. Herein, CRISPR/cas9 genome editing was used to generate a Myc-tagged ATR cell line (mycATR), a heterozygous C-terminal deletion ATR cell line and a tagged at the N-terminal of RPA1 (ATRΔC+/-mycRPA), and a Myc-tagged C-terminal knockout ATR (mycATRΔC). Our findings show that ATR localisation depends upon the C-terminal region and is not limited to the nuclear compartment, but also located to the kinetoplast and in another extra nuclear localisation. The ATRΔC+/-mycRPA cells accumulate single-stranded DNA at the kinetoplast and at the nuclear under or not hydroxyurea treatment. In contrast, we observe abolishment of the possible S-G2/M checkpoint in mycATRΔC cells, leading to aberrant and increased DNA content. However, the complete loss of ATR C-terminal domain did not hinder the replication initiation program. In fact, both cells, mycATR and mycATRΔC cells, use alternative replication sites to maintain the internal chromosomes replication. ATR C-terminal domain is also important to guarantee the replication of the sub-telomeric regions under replicative stress. Finally, complete loss of ATR C-terminal region leads to increased DNA damage and we detect an increased number of aberrant cells accumulating after replication stress exposure. Altogether, the results suggest that the L. major ATR kinase is important during cell cycle control and genome maintenance under replication stress, preventing the accumulation of ssDNA at the nucleus and kinetoplast; as well protecting the replication at sub-telomeric regions. (AU)

• FAPESP's process: 16/16454-9 - Characterization of the ATR kinase of Leishmania major and its role in DNA replication stress signaling
• Grantee: Gabriel Lamak Almeida da Silva
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)