Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

Abstract

DNA polymerase theta (Polθ), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Polθ plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Polθ modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Polθ in different genomic loci; one ortholog is homologous to the Polθ C-terminal polymerase domain, and the other is homologous to the Polθ helicase domain, called Polθ-polymerase and Polθ-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Polθ-helicase from the nuclear extract. Orc1/Cdc6 and Polθ-helicase directly interacted, and Polθ-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Polθ-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Polθ-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.

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

“…However, the PolQ-related protein complement of T. brucei (and, it seems, all kinetoplastids) is highly unusual, since the parasite does not encode a ‘true’ PolQ protein with both DNA Pol and helicase domains, but encodes two related, but discrete DNA Pol and DNA helicase proteins (Figure 1 ). Thus, T. brucei and related kinetoplastids are the only organisms other than metazoans to possess genes encoding separate DNA Pol ( 64 ) and helicase proteins related to PolQ ( 83 ), and the only organisms so far detailed that express only these proteins and not PolQ.…”

“…Again, any link between TbPolIE and the putative kinetoplastid HelQ protein deserves further analysis, since Trypanosoma and Leishmania parasites display robust microhomology-mediated end-joining activity ( 84–86 ), an activity that has so far been ascribed only to PolQ in other eukaryotes ( 49–52 ). Currently, the only work on the putative kinetoplastid HelQ has been in T. cruzi , where the factor has been implicated in DNA replication, not repair ( 83 ). Thus, we do not know if kinetoplastid PolIE and the putative HelQ function together, as has been suggested for PolN and HelQ (Hel308) in human cells ( 58 ).…”

“…First, RNAi depletion of TbPPL2 does not lead to detectable effects on VSG expression (data not shown), which may be because loss of this TLS Pol prevents transition from G2/M into mitosis ( 15 ), unlike the cell cycle perturbations seen after loss of TbPolIE. Second, recent data have implicated HelQ of T. cruzi in DNA replication ( 83 ); if such a role is conserved in T. brucei , and if trypanosomatid HelQ and PolIE proteins work together, then a link between VSG switching and genome replication might be more firmly established.…”

Genome maintenance functions ofTrypanosoma bruceiDNA Polymerase N include telomere association and a role in antigenic variation

“…In this evolutionary context, the PolQ-related protein complement found in T. brucei is highly unusual, since the parasite does not encode a PolQ protein with both DNA Pol and helicase domains, but encodes both PolN-and HelQ-like proteins ( Fig.1). Thus, T. brucei and related trypanosomatids are the only organisms beyond mammals to possess genes encoding both truncated PolQ homologues, and the only organisms so far detailed that express only these proteins and not PolQ [56]. Whether this is because PolQ evolved in some eukaryotes as a gene fusion, or if T. brucei and relatives discarded PolQ is unclear.…”

Gene editing of putative cAMP and Ca2+‐regulated proteins using an efficient cloning‐free CRISPR/Cas9 system in Trypanosoma cruzi

“…Although the CRISPR/Cas9/T7RNAP‐mediated method has been used in T . cruzi (Costa et al, 2018; de Lima et al, 2019; Pavani et al, 2020; Roson et al, 2022), an approach that can be easily scaled up for large‐scale gene editing in this parasite is still needed.…”