Characterisation of XPD from Sulfolobus acidocaldarius : an iron-sulphur cluster containing DNA repair helicase
Abstract
DNA is constantly damaged by a variety of exogenous and endogenous sources. To
maintain the integrity of the genome, different DNA repair mechanisms have evolved,
which deal with different kinds of DNA damage. One of the DNA repair pathways,
Nucleotide Excision Repair (NER), is highly conserved throughout the three kingdoms of
life and deals mainly with lesions arising in the DNA duplex after exposure to UV-light.
The NER pathway in archaea is more closely related to that of eukarya, although the
overall process is not yet well understood. This thesis describes the isolation and
characterisation of one of the repair factors, XPD, from the crenarchaeon Sulfolobus
acidocaldarius (SacXPD).
SacXPD was first identified due to its homology with the eukaryal XPD protein. In
eukarya XPD is the 5a' -> 3a' helicase involved in opening the DNA duplex around a
damaged site. In eukarya, XPD is part of a 10-subunit complex, where it fulfils important
structural roles and takes part in NER, transcription initiation from RNA polymerase II
promoters and cell cycle regulation. The archaeal protein on the contrary is a monomer
and a 5a' -> 3a' SF2 DNA helicase as its eukaryal counterpart. Its cellular functions,
however, are unclear.
Upon purification of SacXPD, it was discovered that the protein binds an ironsulphur
cluster (FeS), which is essential for its helicase activity, but not for any other
enzymatic functions, such as the ATP hydrolysing activity. The FeS cluster domain was
not only identified in archaeal XPD, but also in eukaryal XPD and other related eukaryal
helicases, such as FancJ. The presence of the FeS cluster was confirmed in the eukaryotic
XPD homologue Rad3 from Saccharomyces cerevisiae. Mutagenesis studies were used to
investigate a possible function of the FeS cluster, which may be used to engage ssDNA
during the duplex unwinding process. This would actively distort the ss/ ds DNA
junction. In addition, the resulting bending of the clamped single DNA strand could be
used to avoid reannealing. The consequences of some human mutations introduced into
the SacXPD gene were investigated and could contribute to our understanding of the
development of human diseases.
Type
Thesis, PhD Doctor of Philosophy
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