Identification of proteins involved inTrypanosoma bruceiDNA replication fork dynamics using nascent DNA proteomics

Abstract

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 ...

A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei

“…In Trypanosoma cruzi, FEN-1 participates in DNA replication and repair (66). FEN-1 was also described in an iPOND (isolation of proteins on nascent DNA) MS screen to be associated with replication in T. brucei (67). Furthermore, the spliceosome component Sm-G was enriched with RH2 (68).…”

Profiling the bloodstream form and procyclic form Trypanosoma brucei cell cycle using single-cell transcriptomics

“…Genes relating to ‘ribonucleoprotein complex biogenesis’ include ribosome production factor 2 (RPF2), which is part of the 5S ribonucleoprotein (RNP) complex in PCFs ( Jaremko et al, 2019 ), and 20S-pre-rRNA D-site endonuclease, NOB1, which matures the 3′ end of 18S rRNA ( Kala et al, 2017 ; Figure 5—figure supplement 1 ). “DNA replication” associated genes include replication factors RPA2 and putative, RPC3 ( Figure 5—figure supplement 1 ), both of which show growth defects in PCFs ( Jones et al, 2014 ; Rocha-Granados et al, 2018 ).…”

Profiling the bloodstream form and procyclic formTrypanosoma bruceicell cycle using single cell transcriptomics

“…S10b). “DNA replication” associated genes include replication factors RPA2 and putative, RPC3 (Fig S10c), both of which show growth defects in PCFs 35,50 .…”

“…S10b). "DNA replication" associated genes include replication factors RPA2 and putative, RPC3 (Fig S10c ), both of which show growth defects in PCFs 35,50 . The 1,294 uniquely DE genes in BSFs also showed varied expression dynamics over the cell cycle (Fig.…”