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Mechanisms of how Trypanosoma brucei TRF maintains telomere integrity

Award Number: R21AI169246
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 05/16/2022
PERIOD OF PERFORMANCE END DATE: 04/30/2025

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Mechanisms of how Trypanosoma brucei TRF maintains telomere integrity - Project Summary/Abstract Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are closely related kinetoplastid parasites causing debilitating human diseases. T. brucei sequentially expresses immunologically distinct variant surface glycoproteins (VSGs), its major surface antigen, exclusively from the subtelomeric VSG expression sites (ESs) to evade the host immune response. Similarly, a number of other eukaryotic pathogens that undergo antigenic variation also express their major surface antigens from subtelomeres, and DNA recombination is an important means of antigen switching. Studies of T. brucei telomere biology have shown that perturbation of the telomere structure can be a double-edged sword: increasing telomere stability suppresses VSG switching, while losing gene integrity at the active VSG vicinity results in nearly 90% of cell lethality. We have shown that TRF, the duplex telomere DNA binding factor, plays important roles in maintaining telomere integrity and stability. The active VSG-adjacent telomere is transcribed by RNA Polymerase I into TERRA, which can form the telomeric R-loop (TRL) with the telomeric DNA. TRF suppresses TERRA and TRL levels, and more TRLs in TRF- depleted cells lead to more telomeric DNA damage. However, the underlying mechanisms are unclear. Homologous Recombination (HR) is a major VSG switching pathway, yet deletion of the key HR recombinase RAD51 does not eliminate recombination-mediated switching, indicating that other recombination mechanisms are involved. Microhomology-Mediated End Joining (MMEJ) events exist in T. brucei, but it is unknown whether VSG switching can occur through MMEJ. Antigenic variation is an essential pathogenesis mechanism enabling a long-term parasite infection. Understanding how telomere proteins affect VSG switching and identification of all switching pathways will help us develop means to eradicate this parasite in the future. To better understand how telomere proteins affect VSG switching and to identify additional recombination mechanisms involved in antigenic variation, we will investigate how TRF helps maintain telomere integrity and stability using novel single-molecule analyses – Atomic Force Microscopy imaging (AFM, in air and high-speed in liquids) and DNA tightropes – and genetic and molecular tools through the following aims. In Aim 1, we will examine how TRF suppresses TRL by testing whether TRF binds TRL directly and whether TRF can recruit TERRA to the duplex telomeric DNA by binding to both nucleic acids through different TRF molecules and homodimerization. We will also examine the TERRA localization and R-loop levels in TRF point mutants that weaken or enhance its TERRA binding activity. In Aim 2, we will take advantage that TRF-depleted cells have more recombination products and examine whether HR and MMEJ contribute to telomeric/ subtelomeric instability by deleting/knockdown factors essential for these pathways in TRF RNAi cells. We will then examine VSG switching in cells lacking key recombination players in the WT TRF background. Our studies will reveal how TRF maintains telomere integrity and identify potential additional important factors in antigenic variation.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = -$1,450 )
2025	2023	CLEVELAND STATE UNIVERSITY	2121 EUCLID AVE	CLEVELAND	OH	44115	CUYAHOGA	USA	Allergy and Infectious Diseases Research	000	2	6/17/2025	NON-COMPETING CONTINUATION	-$1,450
														Subtotal = -$1,450

Issue Date FY: 2023 ( Subtotal = $186,500 )
2023	2023	CLEVELAND STATE UNIVERSITY	2121 EUCLID AVE	CLEVELAND	OH	44115	CUYAHOGA	USA	Allergy and Infectious Diseases Research	000	2	4/13/2023	NON-COMPETING CONTINUATION	$186,500
														Subtotal = $186,500

Issue Date FY: 2022 ( Subtotal = $232,737 )
2022	2022	CLEVELAND STATE UNIVERSITY	2121 EUCLID AVE	CLEVELAND	OH	44115	CUYAHOGA	USA	Allergy and Infectious Diseases Research	000	1	5/16/2022	NEW	$232,737
														Subtotal = $232,737

Grand Total All Awards = $417,787

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Mechanisms of how Trypanosoma brucei TRF maintains telomere integrity

Award Number: R21AI169246

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 05/16/2022

PERIOD OF PERFORMANCE END DATE: 04/30/2025

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