View PDF Version - RePub - Erasmus Universiteit Rotterdam

Lesioll demarcatioll
The next step is the formation of an open complex, requiring a local unwinding of
the DNA helix and demarcation of the lesion. XPA has many interactions with other
NER components, for instance with the single strand binding complex RPA [16],
the TFIIH complex [17] and the ERCCIIXPF endonuclease [18] hence XPA may
orchestrate the repair machinery around the DNA lesion. Full opening of the DNA
helix around the lesion is dependent on the presence of ATP [19], strongly arguing
that the helicases of the TFIIH complex (discussed below) are actively involved.
DNA unwinding by TFIIH may be facilitated by RPA, a heterotrimerie complex
involved in NER, replication and recombination [20]. In NER it probably binds to
the single stranded region of the undamaged strand with defined polarity [21]. The
optimal binding patch of RPA is 30 nucleotides [22], which is approximately the
size of the fully opened repair complex and the size of the released damagecontaining
patch. The orchestration of the different NER proteins in the pre-incision
stage ofNER is still unclear.
Tire role of TFIlH ill NER alld basal trallscriptioll
TFIIH is a protein complex of 9 subunits, which was originally identified as an
essential factor in basal transcription initiation [23]. Later, the pS9 and pSO subunits
were rediscovered as the XPB and XPD proteins respectively, involved in NER
[24,25]. They contain ATPase-driven 3'-->5' and 5'-->3' directed DNA heliease
activity respectively, required for local unwinding of the DNA helix around the
lesion in NER [19] and in the transcription initiation of RNA polymerase II at the
promoter [26] (see Figure 2). In accordance with an essential role in basal
transcription, mice with inactivating mutations in the TFIIH subunits XPB and XPD
are inviable (ref. 27 and G. Weeda, unpublished). Other TFIIH components include
Cdk7, cyclin H and MAT!, constituting the cdk-activating kinase (CAK) complex
associated with TFIIH. The CAK complex is able to phosphorylate cyelindependent
kinases (CDKs) involved in cell cycle regulation, and it is required for
phosphorylation of the C-terminal domain of RNA polymerase II [28]. The CAK
complex is more loosely associated with TFIIH and occurs in free form as welL It is
not required for NER in vitro, suggesting that TFIIH can be either in a transcriptionor
in a repair-model [29]. FUlthermore, a functional interaction of TFIIH with p53
has been reported [30,31], suggesting that apoptosis is also associated with TFIIH,
in addition to NER, transcription, and cell cycle regulation.
12 Chapter 1