ATR, a DNA damage kinase, modulates DNA replication timing in Leishmania major

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• Eukaryotic cells employ complex mechanisms to preserve genome integrity against DNA damage and replication stress.
• The protein kinase Ataxia-Telangiectasia and Rad3-related (ATR) is a key regulator of DNA damage response and replication in eukaryotes.
• Leishmania major, a protozoan parasite, possesses a uniquely plastic genome, necessitating investigation into its DNA metabolism regulators.

Purpose of the Study:

• To investigate the functional role of the ATR homolog in Leishmania major's DNA metabolism.
• To determine the impact of ATR C-terminus deletion on DNA replication, cell cycle control, and genome stability in L. major.

Main Methods:

• CRISPR/cas9 genome editing was employed to generate Myc-tagged ATR and C-terminal knockout ATR cell lines (mycATR and mycATRΔC-/-).
• Analysis included assessing nuclear localization, single-stranded DNA accumulation, cell cycle progression, DNA damage levels, and DNA replication restart after stress.

Main Results:

• ATR's nuclear localization is dependent on its C-terminus; deletion led to DNA damage, impaired cell cycle control, and delayed replication restart.
• ATR is vital for L. major's atypical DNA replication program, where larger chromosomes replicate later.
• Loss of ATR's C-terminus caused replication stress accumulation on larger chromosomes, leading to chromosome instability.

Conclusions:

• ATR modulates DNA replication timing in Leishmania major, thereby restricting its genome's plasticity.
• The study highlights ATR's critical role in maintaining genome stability and regulating DNA replication programs in this parasitic protozoan.