Nuclear DNA Replication in Trypanosomatids: There Are No Easy Methods for Solving Difficult Problems

Abstract

In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens.

Keywords: DNA replication; origin recognition complex; replication fork; replication origins; replication stress; trypanosomatid emergence.

Figure 1

Evolutionary Timeline of Eukaryotes. Trypanosomatids diverged around 200–500 million years ago (MYA), probably due to the emergence of new niches precipitated by the appearance of the metazoans, which allowed coevolution of trypanosomatids with both vertebrate and arthropod hosts. Adapted and modified with permission from www.timetree.org.

Figure I

The Main Steps of the Techniques Used to Monitor DNA Replication in Trypanosomatids. DNA combing, MFAseq, and SNSseq. IdU, 5-iodo-2′-deoxyuridine; CldU, 5-chloro-2′-deoxyuridine; PFGE, pulsed-field gel electrophoresis; ssDNA, single-stranded DNA; FACS, fluorescence-activated cell sorting; gDNA, genomic DNA; NGS, next-generation sequencing; T4 PNK, polynucleotide kinase from T4 bacteriophage. Of note, these key steps were described in previous studies , , , , .

Figure 2

The Replication Initiation Machinery of Model Eukaryotes and Trypanosomatids. In model eukaryotes (top panel), from late mitosis to the end of G1 phase, the six-subunit ORC is recruited to all potential origins in the genome. Here, ORC interacts with CDC6. Next, the MCM_2–7 helicase is recruited and loaded onto the origin by Cdt1. This forms the pre-RC, and renders the origins of replication ‘licensed’. At the onset of the S phase, CDC6 and Cdt1 are removed from the pre-RC, CDC45 and GINS are recruited to the origin, forming the pre-IC. Together, CDC45, the MCM_2–7, and the GINS complex form the CMG complex, which is the active replicative helicase that unwinds the origin DNA, allowing the further assembly of the replicative fork components. In trypanosomatids (bottom panel), a divergent ORC-like complex is present, comprising ORC1/CDC6, ORC4, Tb3120, and Tb7980. It is not clear if other subunits remain to be identified. It is assumed that MCM_2–7 is loaded, as in other eukaryotes, prior to the S phase, but how this is catalyzed is unclear, as no Cdt1 orthologue has been identified, and a clear orthologue of CDC6 remains undetermined. It remains possible that MCM is not loaded until the S phase (not shown). At the end of G1 and the start of the S phase, until late G2, an ORC1 orthologue, ORC1B, is expressed. CDC45 and the GINS complex are, most likely, then recruited to the origin, and the steps downstream are believed to take place in a similar way to model eukaryotes. ORC, origin recognition complex; CDC6, cell division cycle subunit 6; MCM, mini chromosome maintenance; Cdt1, CDC-dependent transcript 1; pre-RC, pre-replication complex; CDC45, cell division cycle subunit 45; GINS, ‘go-ichi-ni-san’ in reference to the proteins SId5, Psf1, Psf2, and Psf3; pre-IC, pre-initiation complex; CMG, complex composed of CDC45, MCM_2–7, and GINS.

Figure 3

Replication Fork Progression in Trypanosomatids. All proteins belonging to the replication fork (topoisomerases, MCM_2–7, CDC45, GINS, RPA, PCNA, and Pol α-primase) presented in color have been described in trypanosomatids. Genes encoding homologues of DNA polymerases δ and ε, RFC, and DNA ligase I (presented in gray, with a dashed border) are present in the sequenced genomes of trypanosomatid parasites, but their role in replication fork progression has not yet been characterized. MCM, mini chromosome maintenance; CDC45, cell division cycle subunit 45; GINS, ‘go-ichi-ni-san’ in reference to the proteins SId5, Psf1, Psf2, and Psf3; RPA, replication protein A; PCNA, proliferating cell nuclear antigen; RFC, replication factor C; FEN-1, flap endonuclease 1.

Figure I

The Essential Domains and Motifs of the Main Proteins Involved in DNA Replication in Trypanosomatids. NLS, nuclear localization signal; NES, nuclear export signal; Z, zinc-finger motif; A and B, Walker A and Walker B motifs; R, arginine fingers motif; WHD*, putative winged helix domain; ORC5*, putative ORC5 domain with low significance; ORC2*, putative ORC2 domain with low significance; DHH, (Asp–His–His) motif; Nt and Ct glob, globular domains found at amino and carboxyl terminal regions of the protein; IDCL, inter-domain connecting loop; OBF, oligonucleotide/oligosaccharide-binding fold domain; wHLH, winged-helix-loop-helix domain; L, linkers between OBF domains.

Figure 4

The Replication Stress Response in Mammals and Trypanosomatids. (A) Obstacles that can slow or halt DNA replication leading to replication stress. These include dNTP depletion, DNA lesions (such as thymidine dimers), DNA secondary structures, and DNA:RNA hybrids. In all of these circumstances, the replicative helicase uncouples from the DNA polymerases, causing the accumulation of ssDNA and triggering replication stress-response. (B) The mammalian (top panel) and trypanosomatid (bottom panel) models for the replication stress response. Top panel. In this model, ATR kinase is recruited to RPA-coated ssDNA through its binding partner ATRIP. As an independent event, the 9-1-1 clamp is loaded at the ssDNA–dsDNA junction. TopBP1 is also recruited to RPA–ssDNA at the ssDNA–dsDNA junctions, interacts with the C-terminal tail of Rad9, and stabilizes the ATR kinase localization at the site of stress. ATR phosphorylates downstream factors, including H2A(X) and Chk1, which mediates cell cycle arrest and controls origin firing. Bottom panel. Most of the data for this pathway were observed in Leishmania. These parasites express a functional 9-1-1 homolog; the Rad9 subunit is found in alternative complexes, and Hus1 also exists as a monomer, suggesting a functional flexibility and compartmentalization of the trypanosomatid 9-1-1 clamp. Homologs of key elements of this pathway have not yet been characterized, such as the ATR–ATRIP complex, TOPBP1, or Chk1 (dashed gray molecules). dNTP, deoxynucleotide; ssDNA, single-stranded DNA; ATR, ataxia telangiectasia Rad3-related; RPA, replication protein A; ATRIP, ATR-interacting protein; Rad9, radiation sensitive subtype 9; Rad1, radiation sensitive subtype 1; Hus1, checkpoint protein HUS1; 9-1-1, complex composed of Rad9, Rad1, and Hus1; dsDNA, double-stranded DNA; TopBP1, DNA topoisomerase 2-binding protein 1; H2AX, histone variant; Chk1, checkpoint kinase 1.