Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation

Abstract

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.

TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei

“…Samples were then cooled on ice and the supernatant was harvested and diluted with NuPAGE LDS sample buffer (ThermoFisher) and dithiothreitol to a final concentration of 1× sample buffer with 100 mM dithiothreitol. Harvested proteins were denatured at 70°C for 10 min and then frozen before processing for mass spectrometry using the protocol described in ( 40 ).…”

Trypanosoma bruceiPolIE suppresses telomere recombination

“…Although PolIE was previously identified in a pull-down using a telomere oligo and in a TbTRF IP (48), it has not been directly confirmed that PolIE is a telomere protein (48,49). To verify that PolIE is indeed an intrinsic component of the telomere complex, we first C-terminally tagged one POLIE endogenous allele with 13x myc in the bloodstream form (BF, the mammalian infectious stage) T. brucei cells where one TbTIF2 endogenous allele is C-terminally tagged with an F2H epitope (Fig.…”

“…1A). Among these, DNA polymerase IE (Tb927.11.5550) (48,49) is one of the most abundant proteins. In addition, we have identified TbTRF (36), TbTIF2 (42), TbRAP1 (37), NUP98, NUP96, NUP152, SMC1, SMC3, SMC4, PPL2 (48), and TelAP1 (48), all core histones, H3v, and H2Bv, etc.…”

“…PolIE is an A type DNA polymerase that has a DNA polymerase domain very similar to that in mammalian Polθ (49). However, Polθ has both a C-terminal DNA polymerase domain and an N-terminal helicase domain (50), the latter of which is missing in PolIE (49).…”

“…PolIE is an A type DNA polymerase that has a DNA polymerase domain very similar to that in mammalian Polθ (49). However, Polθ has both a C-terminal DNA polymerase domain and an N-terminal helicase domain (50), the latter of which is missing in PolIE (49). Biochemical studies have shown that Polθ has a weak translesion DNA synthesis activity, while Polθ homologues have been shown to be an important factor of Microhomology-Mediated End Joining (MMEJ) that can help repair DNA double strand breaks (DSBs) (51,52).…”