R-loops acted on by RNase H1 influence DNA replication timing and genome stability in Leishmania

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• Eukaryotic genomes replicate DNA in an ordered manner, creating distinct early and late replicating chromosome regions.
• Leishmania parasites show an unusual chromosome size-dependent DNA replication timing, with larger chromosomes replicating later.

Purpose of the Study:

• To investigate the role of R-loops and RNase H1 in Leishmania's size-dependent DNA replication timing.
• To understand how R-loop resolution impacts genome plasticity and chromosome stability in Leishmania.

Main Methods:

• Analyzing R-loop and RNase H1 accumulation patterns across Leishmania chromosomes.
• Correlating replication timing with chromatin accessibility, G quadruplexes, and sequence content.
• Utilizing conditional gene excision to study the effects of RNase H1 loss.

Main Results:

• R-loops and RNase H1 accumulate in a pattern mirroring replication timing across Leishmania chromosomes.
• Differential organization of R-loops, RNase H1, and replication timing correlates with chromosome size-dependent features.
• Loss of RNase H1 perturbs parasite growth, eliminates size-dependent replication timing differences, and increases aneuploidy and chromosome instability.

Conclusions:

• R-loop homeostasis is intrinsically linked to DNA replication timing in Leishmania.
• The orchestration of DNA replication timing is a key determinant of genome plasticity in this parasitic protozoan.