Summary Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host’s immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homolog has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in-situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.

. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol ␥) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.Trypanosoma brucei and related trypanosomatid parasites (T. cruzi and Leishmania spp.) cause fatal and disfiguring diseases and, subsequently, significant medical and economic stress worldwide, with nearly 500 million people at risk for these vector-borne diseases (7). Current drug treatments are toxic, and no vaccines are available (45). Trypanosomatids are also divergent eukaryotes with a number of unusual biological properties, but one of their most interesting features is their mitochondrial DNA, known as kinetoplast DNA (kDNA). Unlike any DNA structure in nature, kDNA is a network containing thousands of catenated circular DNA molecules (minicircles and maxicircles). Several dozen maxicircles (23 kb) and ϳ5,000 minicircles (1 kb) are condensed into a disk-shaped structure in a specialized region of the cell's single mitochondrion, which is linked to the flagellar basal body through a tripartite attachment complex (16,36,39).The kDNA network is essential for the survival of both procyclic and bloodstream forms of the parasite (42); therefore, understanding kDNA replication and repair processes is an important aspect of trypanosome biology. Network replication is complex, requiring coordinated duplication of each minicircle and maxicircle in near synchrony with nuclear DNA replication (during S phase) (50). Currently, trypanosomatids are the only known eukaryotes to contain at least six mitochondrial DNA polymerases (Pols), namely, two Pol ␤-type enzymes (typically a nuclear repair protein) and four family A Pols related to bacterial DNA Pol I (21, 40). This is in striking contrast to what is the case for other eukaryotes, which contain just one mitochondrial DNA Pol, Pol ␥, for replication and repair transactions.To overcome the topological constraints within the catenated network, a key feature of the replication mechanism is the topoisomerase II-mediated release of individual covalently closed (CC) minicircles into a specialized region called the kinetoflagellar zone (KFZ) (10). Here, the free minicircles initiate unidirectional theta structure replication. Several proteins considered to be involved in this process are also found in th...

29DNA replication, transcription and chromatin remodeling are coordinated to 30 ensure accurate duplication of genetic and epigenetic information. In regard to DNA 31 replication, trypanosomatid parasites such as Trypanosoma brucei display unusual 32 properties including significantly fewer origins of replication than model eukaryotes, a 33 highly divergent Origin Replication Complex (ORC), and an apparent lack of several 34 replication factor homologs. Although recent studies in T. brucei indicate functional links 35 among DNA replication, transcription, and antigenic variation, the underlying 36 mechanisms remain unknown. Here, we adapted an unbiased technology for the 37 identification of replication fork proteins called iPOND (isolation of proteins on nascent 38 DNA) to T. brucei, its first application to a parasite system. This led to the mass 39 spectrometric identification of core replication machinery and of proteins associated with 40 transcription, chromatin organization, and DNA repair that were enriched in the vicinity 41 of an unperturbed active replication fork. Of a total of 410 enriched proteins, among 42 which DNA polymerase  and replication factor C were scoring in the top, around 25%43 of the proteins identified were of unknown function and, therefore, have the potential to 44 be essential trypanosome-specific replication proteins. Initial characterization of a 45 protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of 46 unknown function (Tb927.3.5370) revealed that both proteins retain nuclear localization 47 throughout the cell cycle. While Tb927.3.5370 appeared to be a dispensable gene, 48 Tb927.10.7990 proved to be essential since its silencing caused a growth defect in 49 procyclic cells, accumulation of zoids and impaired DNA replication. Future studies on 50 the generated proteins list can contribute to the understanding of DNA replication 51 dynamics in T. brucei and how replication is coordinated with other cellular processes to 52 maintain genome integrity. 53 54 Introduction 55 Eukaryotic DNA replication is strictly coordinated and regulated by numerous 56 molecular machines to ensure genomic stability for future cell generations. DNA 57 replication initiation is coordinated with cell cycle progression through the multiprotein 58 Origin Recognition Complex (ORC) that plays an essential role by recruiting proteins 59 that lead to the assembly of the replicative machinery with the assistance of regulatory 60 components Cdc6 and Cdt1. The key factors Cdc45, the MCM replicative helicase 61 complex, and GINS proteins form the CMG complex that further recruits other 62 replication factors such as the clamp loader Replication factor C (RFC), the clamp 63 proliferating cell nuclear antigen (PCNA) and the three replicative DNA polymerases 64 (  ) leading to processive DNA replication [1,2]. 65 Instead of the archetypical Origin Recognition Complex (Orc1-6) found in model 66 eukaryotes, trypanosomes contain ORC1 and four other highly divergent ORC subunits ...