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

Loyze P De Lima,Christiane B De Araujo,Leo K Iwai,Elton J R Vasconcelos,M Carolina Elias,Simone G Calderano,Claudio A Pereira,Marcelo S Da Silva,Stenio P Fragoso

doi:10.1038/s41598-019-39348-2

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.

Highlights

DNA polymerase theta (Polθ) is an A family polymerase that functions in genomic maintenance; Polθ has homology to E. coli Pol I1 and is widespread in multicellular eukaryotes but not in fungi[2,3]
origin recognition complex (ORC) is highly divergent from model eukaryotes[15], but we and others have previously shown that Orc1/Cdc[6] is an ORC component that participates in DNA replication[16,17]
Our in silico analysis confirmed the identities of two independent T. cruzi genes (TcCLB.508647.170 and TcCLB.509769.70), which separately encode helicase and polymerase domains, and compared their similarities to genes functionally annotated as Polθ in higher eukaryotes (Fig. 1 and Supplementary Table 1)

Summary

Introduction

DNA polymerase theta (Polθ) is an A family polymerase that functions in genomic maintenance; Polθ has homology to E. coli Pol I1 and is widespread in multicellular eukaryotes but not in fungi[2,3]. Polθ exhibits a C-terminal DNA polymerase domain and an N-terminal helicase-like domain, which has DNA-dependent ATPase activity[11]; a long central domain of unknown function separates these domains. Www.nature.com/scientificreports can join DNA ends with microhomology and mediate the alignment of internal and terminal microhomologous sequences[12] This domain is essential for interstrand DNA crosslink (ICL) repair[11]. The recombinant Polθ-helicase directly interacts with the recombinant TcOrc1/Cdc[6] and is bound to DNA throughout the cell cycle. Our data show that without the polymerase domain, T. cruzi Polθ-helicase directly interacts with Orc1/Cdc[6] and functions as a limiting factor that modulates the binding of MCM to DNA, downregulating replication

Methods

Results

Conclusion