Function of the MRE11 and EXO1 nucleases and BLM helicase in antigenic variation in Trypanosoma brucei

• Grant number: 22/16039-2
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: April 01, 2023
• End date: March 31, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Maria Carolina Quartim Barbosa Elias Sabbaga
• Grantee: Ricardo Obonaga Gómez
• Supervisor: Lucy Glover
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Institution abroad: Institut Pasteur, France
• Associated to the scholarship: 19/01895-8 - Function of the MRE11, DNA2 and EXO1 nucleases in DNA end resection and double-strand breaks repair in Trypanosoma brucei, BP.PD

Abstract

DNA double strand breaks (DSBs) are one of the most toxic forms of DNA damage. These can arise accidentally during normal cell metabolism or after exposure of cells to DNA-damaging agents. Failure to repair them can result in genomic instability, a characteristic of cancer cells. In eukaryotes, DSBs are repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). In HR and microhomology-mediated end joining (MMEJ) (a type of NHEJ Ku heterodimer-independent), the 5´ DNA strands of the DSBs are nucleolytically degraded through a process termed DNA end resection. This process, critical for repair pathway choice by HR or MMEJ and checkpoint signaling, is driven by the MRE11, DNA2 and EXO1 nucleases and generate 32-ended single-stranded DNA tails with different lengths of homology sequences. In most eukaryotes, repair by HR and MMEJ is conserved, including Trypanosoma brucei, the parasite responsible for African human trypanosomiasis, a fatal disease if left untreated. In this parasite, DNA end resection is not well understood. So, in this context, the present research project intends to characterize the functions of the major nucleases in DNA resection and repair of DSBs. Thus, the T. brucei MRE11 and EXO1 nucleases and BLM helicase will be tagged with different epitopes using a CRISPR/Cas9 alternative editing system without selection marker, and then one or two genes will be silenced in different configurations using RNA interference. Instead of DNA2, the BLM gene will be silenced, because we did not find a reliable candidate for the DNA2 protein using sequence alignment tools. Besides that, the choice of studying BLM was made because it works together with DNA2 to resect the DNA end in yeast and mammalian cells. With this approach, we will measure parameters such as nuclear localization, DNA-bound and proteins levels. The DNA end resection will be measured using qPCR from a single DSB generated by the activity of the I-Scel restriction enzyme fused to destabilization domain (DD), which will be controlled in space and time by stabilization with Shield. Additionally, the functions of these nucleases in a DSB repair will be evaluated using a reporter system that allows the reconstitution of green or red fluorescent protein genes depending on the length of the resection. Furthermore, we will determine whether there are differences in the participation of these nucleases in the DNA end resection and repair of DSBs cell cycle dependent. With these results, we hope to contribute with the expansion of our understanding on how the first steps of DNA DSBs repair occur in T. brucei. (AU)