title: Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression.
abstract: The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.
authors.affiliation: a Department of Biochemistry ; Virginia Polytechnic Institute and State University ; Blacksburg , VA USA.
authors.forename: Ana L
authors.initials: AL
authors.lastname: Valenciano
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Ramsey
authors.forename: Zachary B
authors.initials: ZB
authors.lastname: Mackey
mesh_terms: D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000502:physiology*; D005434:Flow Cytometry; D059565:G2 Phase Cell Cycle Checkpoints / Q000502:physiology*; D015151:Immunoblotting; D008156:Luciferases; D008856:Microscopy, Fluorescence; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014346:Trypanosoma brucei brucei / Q000378:metabolism / Q000502:physiology*
chemical_list: D017931:DNA Primers; D018809:Proliferating Cell Nuclear Antigen; D008156:Luciferases
keywords: CDK, cyclin dependent kinase; Cd, Cluster of differentiation; DAPI, 4′, 6-diamidino-2-phenylindole; DNA replication; EdU, 5-Ethynyl-2′deoxyuridine; GINS, Go, Ichi, Nii, complex; Gadd, growth arrest and DNA-damage; H2O2, hydrogen peroxide; HU, hydroxyurea; Hs, Homo sapiens; Mcm, mini-chromosome maintenance proteins; MyD, myeloid differentiation primary response gene; Orc, origin recognition complex; PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcriptase-polymerase chain reaction; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tb, Trypanosoma brucei; attenuate; chemosensitize; hydroxyurea; proliferation