Date of Completion

5-27-2015

Embargo Period

5-20-2015

Keywords

Trypanosoma brucei, cyclin dependent kinase, CDK, parasitology, DNA repair, TFIIH, XPB, Nucleotide Excision Repair

Major Advisor

Dr. Arthur Günzl

Associate Advisor

Dr. Asis Das

Associate Advisor

Dr. Blanka Rogina

Associate Advisor

Dr. Bruce Mayer

Associate Advisor

Dr. Gordon Carmichael

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Eukaryotic TFIIH consists of a core of seven subunits, including the DNA helicase Xeroderma Pigmentosum B (XPB), and a cyclin-dependent kinase (CDK)-activating complex (CAK) that contains CDK7. XPB is crucial for DNA unwinding during transcription and nucleotide excision repair (NER) while the CAK complex phosphorylates RNA polymerase II (RNAPII) carboxy terminal domain (CTD), enabling the enzyme’s promoter clearance.

Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are lethal human parasites, belonging to the early-diverged order Kinetoplastida. They transcribe genes polycistronically and require spliced leader (SL) trans splicing for the maturation of mRNAs from polycistronic precursors. SL RNA gene (SLRNA) transcription by RNAPII depends on trypanosome TFIIH complex which contains orthologs of all core subunits but lacks the CAK complex. Despite this, the trypanosome CTD is phosphorylated and essential for SLRNA transcription. Gene silencing of CDC2-related kinase 9 (CRK9) revealed its importance for parasite survival and CTD phosphorylation. Interestingly, this loss of phosphorylation did not cause a specific RNAPII transcription defect. Instead, CRK9 silencing blocked trans splicing and caused hypomethylation of SL RNA’s extensively modified cap. Sedimentation analysis of tandem affinity purified CRK9 revealed a putative tripartite complex including a novel L-type cyclin (CYC12), and a CRK9-associated protein (CRK9AP). Silencing CYC12 or CRK9AP recapitulated the defects observed upon CRK9 silencing, confirming that they functionally partner with CRK9 in vivo. CRK9AP depletion caused a rapid co-loss of CRK9 and CYC12, suggesting its role in complex assembly. This project identified CRK9 as crucial for parasite-specific gene expression. As a CDK, CRK9 is a promising drug target against trypanosomes which was validated in a mouse model.

Another feature related to trypanosome TFIIH is the presence of two divergent paralogs of XPB in kinetoplastid genomes while only the larger XPB paralog consistently co-purified with TFIIH. Gene knockout of the second paralog, termed XPB-R showed that XPB-R is specialized in NER but dispensable for transcription. While XPB-R does not assemble into a TFIIH complex, reciprocal co-immunoprecipitations revealed an interaction with the p52 ortholog, a TFIIH component and known regulator of XPB in other systems, indicating that trypanosomes possess a TFIIH whose function is restricted to transcription and a XPB-R/p52 repair complex.

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