Institute of Biomedical Sciences

Title

Analysis of DNA break repair inhibition in Trypanosoma brucei

Document Type

Poster

Abstract Category

Immunology/Infectious Diseases

Keywords

Trypanosoma brucei, DNA break repair, antigenic variation

Publication Date

Spring 5-1-2019

Abstract

African trypanosomes (Trypanosoma brucei spp.), the cause of African Sleeping sickness, are masters of antigenic variation and can change their dense variant surface glycoprotein (VSG) coat to new variants, thereby escaping host immunity. Switching VSG gene expression is thought to occur predominantly following DNA break formation at naturally unstable telomeric ends. Trypanosomes appear to not have a classical non-homologous end-joining pathway (NHEJ), which predominates in mammalian DNA double-stranded break (DSB) repair. Rather, DSB repair in T. brucei occurs through homologous recombination (HR) and microhomology mediated end-joining (MMEJ). Mechanisms of HR predominate in DSB repair and are currently the only known mechanisms that can result in the activation of a new VSG gene, a process called “VSG switching”. Thus, tracking DNA break formation events that occur can elucidate whether the break will be repaired in a manner that results in blunt end-joining or the expression of a new VSG through HR. While T. brucei DNA break repair pathways are only partially known, the factors that regulate the outcome of a DNA break repair are unknown. Here, we investigate the consequences of inhibiting two subsequent steps of HR, end resection by MRE11 and the coating of single-stranded DNA filaments with RAD51, which then promotes homologous pairing. We sought to determine if known chemical inhibitors of mammalian DSB repair, developed in cancer research, function in T. brucei based on a collection of phenotypes, both previously published and novel to this investigation. We determined that both RAD51 inhibitor RI-1 and the MRE11 inhibitor mirin, have the anticipated effects on cell growth and sensitivity to DNA damage. However, we also observed distinct behaviors of each inhibitor on both the processing of DNA breaks and the progression of cell cycle. These data further support previous T. brucei literature that suggests MRE11 does not exclusively function upstream of RAD51. Furthermore, our findings indicate both MRE11 and RAD51 may integrate signals for non-canonical processes of DNA break repair and cell cycle progression checkpoints, whose identification could yield novel drug targets against African trypanosomiasis. Chemical inhibitors thus provide a new avenue for further studying the factors regulating VSG switching in T. brucei.

Open Access

1

Comments

Presented at Research Days 2019.

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Analysis of DNA break repair inhibition in Trypanosoma brucei

African trypanosomes (Trypanosoma brucei spp.), the cause of African Sleeping sickness, are masters of antigenic variation and can change their dense variant surface glycoprotein (VSG) coat to new variants, thereby escaping host immunity. Switching VSG gene expression is thought to occur predominantly following DNA break formation at naturally unstable telomeric ends. Trypanosomes appear to not have a classical non-homologous end-joining pathway (NHEJ), which predominates in mammalian DNA double-stranded break (DSB) repair. Rather, DSB repair in T. brucei occurs through homologous recombination (HR) and microhomology mediated end-joining (MMEJ). Mechanisms of HR predominate in DSB repair and are currently the only known mechanisms that can result in the activation of a new VSG gene, a process called “VSG switching”. Thus, tracking DNA break formation events that occur can elucidate whether the break will be repaired in a manner that results in blunt end-joining or the expression of a new VSG through HR. While T. brucei DNA break repair pathways are only partially known, the factors that regulate the outcome of a DNA break repair are unknown. Here, we investigate the consequences of inhibiting two subsequent steps of HR, end resection by MRE11 and the coating of single-stranded DNA filaments with RAD51, which then promotes homologous pairing. We sought to determine if known chemical inhibitors of mammalian DSB repair, developed in cancer research, function in T. brucei based on a collection of phenotypes, both previously published and novel to this investigation. We determined that both RAD51 inhibitor RI-1 and the MRE11 inhibitor mirin, have the anticipated effects on cell growth and sensitivity to DNA damage. However, we also observed distinct behaviors of each inhibitor on both the processing of DNA breaks and the progression of cell cycle. These data further support previous T. brucei literature that suggests MRE11 does not exclusively function upstream of RAD51. Furthermore, our findings indicate both MRE11 and RAD51 may integrate signals for non-canonical processes of DNA break repair and cell cycle progression checkpoints, whose identification could yield novel drug targets against African trypanosomiasis. Chemical inhibitors thus provide a new avenue for further studying the factors regulating VSG switching in T. brucei.