Building a molecular machine : heterologous expression and biochemical characterisation of transcription factor IIH

Abstract

Organisms across Archaea, Bacteria and Eukarya possess a series of mechanisms to protect the integrity of the deoxyribonucleic acid (DNA) from the constant attack of internal and external factors that modify the structure of the double helix, potentially causing an increase in mutagenesis. The Nucleotide Excision Repair (NER) pathway removes bulky lesions in the DNA caused by ultraviolet (UV) radiation and other sources that can introduce a strong distortion in the double helix. A key element in this repair mechanism is the transcription factor IIH (TFIIH), a ten-subunit complex which opens and extends the DNA bubble originated at the damaged site to allow the subsequent repair factors access to the lesion, so the insult can be removed. Mutations in TFIIH subunits xeroderma pigmentosum group B (XPB), xeroderma pigmentosum group D (XPD) or p8 can cause a series of autosomal recessive disorders with symptoms that include mild-to-extreme photosensitivity, progeria, physical and neurological abnormalities, and in some cases an increased susceptibility to cancer. This thesis describes the processes of cloning, expression and purification followed to obtain damage-detector heterodimer XPC-HR23B, the 7-subunit TFIIH Core sub-complex and the 10-subunit TFIIH complex by means of a powerful baculoviral expression vector (BEV) called MultiBacTM, a tool specifically conceived for the obtaining of multi-subunit eukaryotic complexes. The biochemical characterization of TFIIH Core showed that the sub-complex can bind a series of double-stranded DNA (dsDNA) substrates with an affinity in the nM range, and the presence of a bubble or damage at the duplex does not change TFIIH Core’s binding affinity significantly. The study of TFIIH Core’s unwinding ability confirmed that the sub-complex can open a series of dsDNA substrates only when a 5’ overhang end is available, and this activity can be stalled by a lesion located in both the translocating and non-translocating strands.