Identification of proteins involved in Trypanosoma brucei DNA replication fork dynamics using nascent DNA proteomics

DNA replication, transcription and chromatin remodeling are coordinated to ensure accurate duplication of genetic and epigenetic information. In regard to DNA replication, trypanosomatid parasites such as Trypanosoma brucei display unusual properties including significantly fewer origins of replication than model eukaryotes, a highly divergent Origin Replication Complex (ORC), and an apparent lack of several replication factor homologs. Although recent studies in T. brucei indicate functional links among DNA replication, transcription, and antigenic variation, the underlying mechanisms remain unknown. Here, we adapted an unbiased technology for the identification of replication fork proteins called iPOND (isolation of proteins on nascent DNA) to T. brucei, its first application to a parasite system. This led to the mass spectrometric identification of core replication machinery and of proteins associated with transcription, chromatin organization, and DNA repair that were enriched in the vicinity of an unperturbed active replication fork. Of a total of 410 enriched proteins, among which DNA polymerase α and replication factor C were scoring in the top, around 25% of the proteins identified were of unknown function and, therefore, have the potential to be essential trypanosome-specific replication proteins. Initial characterization of a protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of unknown function (Tb927.3.5370) revealed that both proteins retain nuclear localization throughout the cell cycle. While Tb927.3.5370 appeared to be a dispensable gene, Tb927.10.7990 proved to be essential since its silencing caused a growth defect in procyclic cells, accumulation of zoids and impaired DNA replication. Future studies on the generated proteins list can contribute to the understanding of DNA replication dynamics in T. brucei and how replication is coordinated with other cellular processes to maintain genome integrity.

143 splicing, and chromatin organization factors. Nearly 25% of the data set were proteins of 144 unknown function. Overall, we obtained a panoramic view of the cellular processes that 145 appear to be coordinated with DNA replication and might help to maintain genomic 146 stability. Additionally, we selected two proteins for initial characterization. These are a 147 putative Replication Factor C subunit (RFC) and a protein of unknown function. Both 148 proteins displayed nuclear localization. Only RFC proved to be essential in PCF cells.
149 Cells in which this protein was depleted exhibited a DNA replication defect and growth 150 impairment.

152 Materials and Methods
153 For Primer sequences refer to Supplemental Table 1 (S1 Table). 282 Image slices were reconstructed using default software parameters and 3D 283 deconvolution using the automatic method in NSIM modality was applied. Images' 284 brightness and contrast were adjusted using Adobe Photoshop CS4 for presentation in 285 figures. 299 (ii) EdU Fluorescence Intensity. Images were acquired using the Nikon E600/Spot 300 digital camera system (described above). Non-saturating exposure times were used and 301 non-adjusted images were analyzed using CellProfiler 3.0.0  Table).

381
To include proteins in our list that were identified in our nascent DNA analysis but   518 discrete spots that likely represent replication foci (Fig 1B).

519
The iPOND technology relies on click chemistry, which is a copper-catalyzed 520 reaction that allows the cycloaddition of an alkyne functional group (present in EdU) to   540 experiments, 1.76 pmoles (0.12 µg of DNA) of biotinylated DNA was detected from the 541 2 µg of DNA that was spotted, while there was no detection in the negative controls (Fig   542 1C). Biotinylated DNA was also detected in the thymidine chase experiment (S1E Fig). 543 These modifications were critical to achieve approximately 24 µg of EdU-labeled DNA in 544 a 10 min EdU pulse, comparable to the 28 µg regularly obtained in mammalian iPOND.
545 In a 10 min pulse, we should label ~37 kbp. (Table 1). See S1 Appendix for details on 546 calculations for iPOND.

562
To test whether unmodified or K76-methylated histone H3 was present in our 563 final iPOND eluates we carried out western blot analysis using T. brucei specific 564 immune sera. There is minor detection of H3 in the EdU sample while its signal 565 increases in the ThD chase sample (Fig 1E, S2 Fig). Methylated H3 is mainly detected 566 in the ThD chase sample (Fig. 1E). For some EdU samples minor amounts of 567 H3K76me1 can be detected (Fig. 1E), however methylated H3 was rarely detected in 568 the EdU samples (S2 Fig). 569 Quantification of band intensities revealed a 1.5% recovery of total H3 signal in 570 the EdU elution compared to its input signal. In contrast, 13% of the H3 signal was

597
Based on these robust criteria, a total of 410 proteins were found to be enriched 598 on nascent DNA (S2A Table). The genes of 98 proteins were annotated as "hypothetical 599 conserved", encoding proteins of unknown function (S2B Table). Gene Ontology (GO) 600 enrichment analysis using the tool PANTHER [49] revealed 23 GO terms with >3-fold 601 enrichment and a P-value of <0.001 (Fig 2A, S3 Table). 610 and the DNA repair and nucleic acid metabolism clusters. However, there were also 611 abundant links between DNA replication and transcription clusters (Fig 2B).

917
In addition, our data set has provided a list of proteins of unknown function that 918 can be characterized to determine their function in DNA replication and/or at nascent 919 DNA. They could represent essential trypanosomatid-specific factors or extremely 920 divergent homologues of known replication factors and, therefore, represent promising 921 targets of chemotherapeutic intervention. The initial characterization of the protein 922 candidate Tb7990, a replication factor C subunit, indicated an essential role in DNA 923 replication that was quantified using EdU incorporation and microscopy assays.
924 However, many of the DNA replication proteins identified in our data set have not been 925 studied and their future characterization using our strategy to detect replication defects