DNA replication and repair represent two evolutionarily conserved DNA metabolism pathways that function to maintain genomic integrity in all kingdoms. These pathways in kinetoplastid parasites are fascinating because they contain rapidly evolving genes with atypical biological and biochemical properties that contrast to properties of their mammalian host orthologs. Our long-term goal is to understand the molecular anatomy of essential genes that guard genomic integrity in BSF Trypanosoma brucei. Understanding T. brucei DNA replication and repair pathways down to the mechanistic detail has valuable implications for mutagenesis and carcinogenesis. Essential DNA replication or repair pathway members with atypical properties represent targets that can be harvested as “low hanging fruit” for parasite-specific therapies.
Identifying proteins in the replisome of Trypanosoma brucei.
Previous studies have shown that the parasite does use a homolog of the Proliferating Cell Nuclear Antigen (PCNA) and extracellular-signal regulated kinase 8 (ERK8). However, the remainder of the replisome is less characterized. Our goal is to demonstrate a more comprehensive view of the T. brucei replisome to identify potential therapeutic targets.
Probing structural integrity in the Proliferating Cell Nuclear Antigen of Trypanosoma brucei (TbPCNA)
Alignment of TbPCNA with homologs from human shows an amino acid insertion from residues 186 to 226 that is predicted to form a unique backside loop, shown below. This loop contains a reactive cysteine (C210) that is predicted to be a the base of a small pocket. Mutation of this cysteine to a serine (C210S) has produced preliminary data that shows extensive oligomerization of TBPCNA in vitro. Research into this reactive cysteine may help validate this as a potential therapeutic target.
Understanding how TbERK8 regulates DNA replication in Bloodstream Form (BSF) T. brucei.
We had identified TbERK8, an essential extracellular-signal regulated kinase (ERK) family member. ERKs are serine/threonine-specific protein kinases that direct cellular responses to a diverse array of external signals. They regulate proliferation, gene expression, differentiation, mitosis, cell survival, apoptosis and are implicated in DNA damage response. We demonstrate that silencing TbERK8 in BSF T. brucei causes replication arrest, severe nuclear fragmentation and stage-specific lethality in the parasite. No lethal consequence was reported when others had silenced the ERK8 homolog in human cells. ERK8 deficient human cell show decreased levels of replication and increased levels of DNA strand breaks, indicating that the influence of ERK8 homologs on DNA replication and repair is conserved across divergent eukaryotic species. Our results demonstrate that the lethal effect of silencing TbERK8 is specific to BSF T. brucei.