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

ABSTRACT

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