TFIIH
* Discovery of TFIIH
In the next year after identification of TFIIF in 1990, Flores
and his colleagues separated transcription factor IIH by chromatography on
a phenyl-Superose from TFIIF. It was initially identified as a general transcription
factor required for basal transcription. However, some of the outstanding
breakthroughs have also been made when some new and unexpected role played
by a known protein is rediscovered. In 1993, Schaeffer and co-workers found
that XPB, a subunit of TFIIH, is also a repair factor. In the same year, Kevin
S. Sweder proposed that the ERCC3 has different roles in transcription initiation
and NER.
TFIIH
TFIIH, a nine-subunit protein complex, plays a dual role in both
DNA excision repair and in transcription. Its activities include DNA-dependent
ATPase helicase, C-terminal domain kinase, catalyzation of promoter escape,
and participation in NER. During the transcription, TFIIH enters the RNA polymerase
II preinitiation complex and activates it prior to initiation. It may act
in open complex formation and/or promoter clearance. In NER, TFIIH is recruited
by XPA to the damaged sites (Park et al., 1995). At least two excision-repair
proteins, encoded by the xeroderma pigmentosum (XPB/ERRC3) and XPD/ERR2 subunits,were
found in TFIIH (Drapkin et al., 1994). This suggested that the TFIIH complex
might involve in excision repair. During NER, TFIIH is thought to convert
a damaged site into a substrate for the XPF and XPG. Both in NER and transcription
initiation, TFIIH is required for melting the DNA double helix (Weaver, 2001). Maintenance
and melting of this open complex need the helicases of TFIIH (Okhuma, 1997). In addition
to unwinding, TFIIH has other functions in NER. Evans (1997) found that TFIIH
facilitates the incision by XPF and the interaction with CSA may be relevant
for TCR (Henning et al., 1995). The double life of TFIIH is fulfilled through
its nine subunits, designated XPB/ERCC3, XPD/ERCC2, GTF2H1/p62, GTF2H4/p52,
GTF2H2/p44, GTF2H3/p34, CDK7, CCNH, and MNAT1. The subunits of XPB and XPD
have DNA-dependent ATPase and helicase function. A subcomplex, containing
CDK7, CCNH, and MNAT1, phosphorylates the carboxy-terminal domain (CTD),
as well as several cyclin-dependent kinases (cdks). The GTF2H2 (p44) and
GTF2H3 (p34) subunits, containing zinc finger motifs, are believed to have
DNA-binding capacity. As yet, no specific functions have been found for the
remaining GTF2H4 (p52) and GTF2H1 (p62).
Subunits
XPB
XPB/ERCC3, a helicase, is the largest subunit of TFIIH. The XPB
is about 89K polypeptide at 2q21 of gene location. It’s homolog to RAD25 in
S. Cere. Mutation in XPB is associated with DNA-repair disorders xeroderma
pigmentosum (XP) and Cockayne’s syndrome. Using recombinant wild type or mutated
XPB (p 89k) polypeptides and different forms of DNA template, there was a
connection found between the ATPase and the helicase (R. Roy, 1994). The
helicase with a direction of 3’-5’, and a DNA-dependent ATPase are associated
with TFIIH and reside with the XPB subunit. The nucleotide competition experiment
clearly distinguished ATPase/helicase from the CTD kinase. Addition of antibodies
against XPB to the transcription system resulted in a specific inhibition
of transcription. In the presence of ATP, the helicase activities of XPB along
with XPD unwind the DNA duplex at the start site of transcription or the
preincision intermediate. The ERCC3 may have separate roles in transcription
and NER. Its roles probably depend on the proteins associated with it at that
time. Separation of ERCC3 from the transcription initiation complex may free
the protein to associate with repair enzymes to enable damage recognition
(Sweder et al., 1993)
XPD
The XPD/ERCC2 subunit, the human homologue of yeast RAD3, is
about 80 KD. The XPD gene maps to the long arm of chromosome band 19q13.2-q13.3.
Mutation in XPD can result in genetically heterogeneous disorder, namely xeroderma
pigmentosum complementatin group D (XPD), trichothiodystrophy (TTD), and
Cockayne’s syndrome. In the presence of ATP, the helicase activities of XPD
and XPB subunits unwind the DNA duplex at the start site of transcription
or the preincision intermediate. Although both XPD and XPB are ATP-dependent
helicase subunits, XPD has a 5’ to 3’ DNA helicase, which is opposite to XPB.
Besides TFIIH, XPD is also associated with CAK complex (Rossignol et al.,
1997). This is required for transcription initiation but is dispensable
for in vitro NER (Sung et al., 1996). Addition of antibodies against
XPD to the transcription system resulted in a specific inhibition of transcription
(Rossignol, et al., 1997)..
GTF2H1/p62 Core TFIIH subunit
The GTF2H1 is polypeptide of 548 amino acids with a molecular
weight of 62 kD and an isoelectric point of 8.82. It is homolg to TFB1 in
S. cere and its gene is located at 11p14-15.1. No kinase motifs or DNA binding
motifs had been found in FTF2H1 (Fischer, 1992). However, a molecular complex
containing the XPB and p62 subunits can interact with CSB, which in turn interact
with polymerase II (Tantin, 1998). When over expressed in Escherichia coli
or in insect cells, the recombinant GTF2H1 had the same electrophoretic mobility
on SDS-PAGE as the one from BTF2. A monoclonal antibody to GTF2H1 can inhibit
transcription in vitro. Immunoaffinity experiments showed that GTF2H1 is
closely associated with the other subunits present in the TFIIH (Fischer
et al., 1992).
GTF2H2/p44 Core TFIIH subunit
GTF2H2 subunit has a molecular weight of 44KD. The gene encoding
GTF2H2 located at 5q12.2-q13.3 appeared to be the human counterpart of SSL1,
a gene involved in translation and UV resistance in yeast. GTF2H2 containing
zinc fingers may play a role in DNA binding (Humbert, 1994). It has been reported
by Burglen (1997) that the gene encoding GTF2H2 is involved in Werdnig-Hoffmann
disease.
GTF2H3/p34 Core TFIIH subunit
The gene encoding GTF2H3, a subunit of the basal transcription
factor TFIIH with a molecular weight of 34KD, has been characterized and localized
in the 12q region. Similar to GTF2H2, GTF2H3 also has homology with
a domain of SSL1 in yeast. Both GTF2H2 and GTF2H3 possess zinc finger domains
that may mediate BTF2 binding to DNA (Humbert, 1994).
GTF2H4/p52 Core TFIIH subunit
GTF2H4, with a molecular weight of 52 KD, is homolg to TFB2 in
S. cere. Its gene is located at 6p21.3 region. So far, no activity has been
found for this subunit.
CDK7 Kinase subunit of TFIIH
CDK7, the human homologue of yeast KIN28, has a molecular weight
of 41KD. Its gene is located at 2p15-cen. Cyclin-dependent kinase (CDK) plays
an essential role in cell cycle. The activation of CDK requires phosphorylation
of a conserved residue of a CDK by the CDK-activating kinase (CAK). CAK in
mammalian cells is composed of two key proteins: CDK7 and CCNH. CDK7-CCNH
assembly requires either phosphorylation of CDK7 or MNAT1 (Devault et
al., 1995). CDK7 phosphorylates the CTD of RNA polymerase II during transcription
(Svejstrup, 1996). This phosphorylation of the CTD is believed to induce the
transition from initiation to elongation (Usheva et al., 1992). Together
with the CCNH and MNAT1 subunits of TFIIH, CDK7 is probably involved in cell
cycle regulation (Rossignol, 1997). There is a contradiction concerning whether
CDK7 is required for the repair activity. Roy (1994) believed that CDK7 was
essential for excision repair. On the contrary, David Mu (1996) came up an
opposite conclusion that the CDK7-cyclin H pair neither is necessary for nor
interferes with the repair function of TFIIH in human. In addition, CDK7 does
not dissociate from holo-TFIIH during the NER, according to the strong interaction
of CDK7 with the core subunits of TFIIH.
CCNH Kinase subunit of TFIIH
CCNH, the human homologue of yeast CCL1, has a molecular weight
of 38 KD and its gene located at 5q13.3-q14. It has cyclin activity. CCNH,
CDK7, and MNAT1 subunits comprise the subcomplex that phosphorylates the CTD
of RNA polymerase II during transcription (Svejstrup, 1996). It may be also
involved in cell cycle regulation (Rossignol, 1997).
MNAT1 Kinase subunit
Its gene is located at 14q23. MNAT1 may increase the efficiency
of CDK7-CCNH assembly (Devault, 1995). The CDK7-CCNH-MNAT1 ternary complex
phosphorylates the CTD of RNA polymerase II, which is both required for transcription
(Svejstrup, 1996) and for DNA nucletide excision repair. It may be also involved
in cell cycle regulation (Rossignol, 1997).