Molecular & Biochemical Parasitology 193 (2014) 110–113 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology Short communication ORC1/CDC6 and MCM7 distinct associate with chromatin through Trypanosoma cruzi life cycle Simone Calderano a,c , Patricia Godoy a , Daiane Soares a,c , Osvaldo Augusto Sant’Anna b,c , Sergio Schenkman d , M. Carolina Elias a,c,∗ a Laboratório Especial de Ciclo Celular, Instituto Butantan, São Paulo, Brazil b Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil c Center of Toxins, Immune Response and Cell Signaling – CeTICS, Brazil d Departamento de Micro, Imuno e Parasitologia, Universidade Federal de São Paulo, Brazil a r t i c l e i n f o Article history: Received 27 December 2013 Received in revised form 17 February 2014 Accepted 17 March 2014 Available online 25 March 2014 Keywords: DNA replication ORC1/CDC6 MCM7 Trypanosoma cruzi a b s t r a c t Trypanosoma cruzi alternates between replicative and non-replicative stages. We analyzed the expression of components of the pre-replication machinery TcORC1/CDC6 and TcMCM7 and their interaction with DNA in all T. cruzi stages. TcORC1/CDC6 remains in the nuclear space during all stages of the life cycle and interacts with DNA in the replicative stages; however, it does not bind to DNA in the non-replicative forms. Moreover, TcMCM7 is not present in the non-replicative stages. These data suggest that the lacking of DNA replication during the T. cruzi life cycle may be a consequence of the blocking of TcORC1/CDC6–DNA interaction and of the down regulation of the TcMCM7 expression. © 2014 Elsevier B.V. All rights reserved. Trypanosoma cruzi, the etiological agent of Chagas’ disease, is an early divergent protozoon eukaryote. During its life cycle, it alternates between replicative and non-replicative stages in insect and mammalian hosts. In the insect vector, the replicative epi- mastigote stage differentiates into the non-replicative/infective metacyclic trypomastigote form, and in mammalian hosts, the replicative amastigote differentiates into non-replicative/infective trypomastigote stage. Although T. cruzi has been extensively stud- ied since its discovery more than one century ago, the molecular basis for the lack of DNA replication in non-replicative forms remain to be determined. DNA replication is initiated with the assembly of the pre- replication complex (PRC) at the origins of replication. In eukaryotes, the PRC is composed of an ORC (origin recog- nition complex), a six-subunit complex (Orc1-6) that recruits Cdc6 to the replication origin in late M-phase, and the MCM (mini-chromosome maintenance) helicase, which is comprised of Mcm2-7 and is loaded by the ORC–Cdc6 complex with Cdt1 onto DNA [1]. During this loading reaction that occurs during the G1 stage, Cdt1 is released from the DNA while MCM encircles ∗ Corresponding author at: Laboratório Especial de Ciclo Celular, Intituto Butantan, São Paulo, Brazil. Tel.: +55 11 3726 1024; fax: +55 11 3726 1024. E-mail address: carolina.eliassabbaga@butantan.gov.br (M.C. Elias). double-stranded DNA [2,3]. During S-phase, the MCM helicase is activated by the interaction with Cdc45 and the GINS (from the Japanese go-ichi-ni-san) complex. The assembly of Cdc45 and GINS converts the PRC into a pre-initiation complex. The origins of repli- cation are unwound, and the replicative DNA polymerases as well as the regulatory factors of the replication machinery are recruited, which allows for the establishment of replication forks [1]. The blockage of DNA replication has been studied in detail during the cell cycle, which is when re-replication might be avoided during and after S phase. As the PRC determines the sites where replica- tion will occur, an effective method to block DNA re-replication is through inactivating the pre-replication machinery or by down reg- ulating its expression or activity when DNA replication is initiated. The inactivation of the PRC during and after S phase occurs through different mechanisms in different organisms. In yeast, the ORC sub- units are bound to chromatin throughout the cell cycle; however, in mammalian cells, the Orc1 subunit is degraded in S phase by a CDK-dependent polyubiquitination reaction [4], and phosphoryla- tion of Orc2 dissociates the ORC from chromatin [5]. In yeast, Cdc6 is phosphorylated by cyclin-dependent kinases (CDK) and degraded by proteolysis at the onset of the G1-S phase transition, whereas mammalian Cdc6 remains bound to chromatin throughout the cell cycle. In addition, MCM are exported from the nucleus during S phase, G2, and early mitosis in yeast, which prevents the licensing of new origins in non S-phase stages (reviewed in [6]). http://dx.doi.org/10.1016/j.molbiopara.2014.03.004 0166-6851/© 2014 Elsevier B.V. All rights reserved.