Results p resen ted so far suggest th at m ost TFIIH com plexes w ith m utations in XPD leading to XP features are defective in NER. The high levels of UDS show n by these cells w ere therefore n o t due to specific placem ent of the two NER incisions around the DNA lesion. If one assum es that the assem bly of the NER m achinery on a DNA lesion is affected by the XPD inactivating m utation, this could lead to som e extent of defective op ening d u rin g the NER reaction an d to the placem en t of only the 5'
incision. This w ould then provide a free 3'-O H group at w hich the DNA replication m achinery could initiate DNA synthesis. The relatively high level of dam age-dependent unscheduled DNA synthesis observed in vivo
m ight therefore be due to aberrant synthesis of DN A fragm ents different than 26-30 nucleotides (that are generated in the w ild type NER reaction), after 5' unco u p led incisions are m ade. H ow ever, n eith er open complex form ation no r un co u p led 3' incisions w ere ever d etected in vitro using w hole cell extracts from XP-D cells of patients XP7BE and XP17BE (R683W am ino acid substitution) (Evans et al. 1997b).
U ncoupled 3' incisions have been rep o rted to be m ade by one of the TFIIH m utants. Cells from patien t XP11BE have TFIIH containing an XPB subunit w hich is the product of aberrant splicing (Weeda et a l 1990; H w ang
et al. 1996). This alteration of the C -term inus of the protein does not inhibit 3' incisions b u t prevents form ation of 5' incisions (Evans et a l 1997b). 3' u n co u p led incisions are also form ed in XPF- an d E R C C l-defective cell extracts (Sijbers et a l 1996a). This suggests th at XPB helicase m ight be in contact w ith or m odulate the activity of the 3' nuclease ERCC1-XPF (Evans
et a l 1997b). U ncoupled 5' incisions have also been reported. Site-directed
m u ta tio n s in activ atin g the catalytic site of XPG e n d o n u clea se allow form ation of 5' incisions w ith o u t the concom itant 3' incision (C onstantinou
et a l 1999). No naturally occurring XPG m utations of this type are known. 145
C h ap ter V - XPD m utants W o u ld the XP-D cell extracts p e rfo rm 5' u n c o u p le d incisions a n alo g o u s
to the w ay XP11BE does the 3 ? In S . c e r e v i s i a e , a low level of n ick in g of UV-
irra d ia te d D N A is o b serv ed w h en Rad3 A rg-48 m u ta n t p ro te in is te ste d in a
re c o n stitu te d NER incision assay u sin g U V -d am ag ed D N A , b u t no excised
frag m e n ts are sim u ltan eo u sly d e te c ted (Sung et al. 1988; S ung et al. 1996). To
e sta b lish w h e th e r XPD m u ta n t TFIIH fu n c tio n s in th e fo rm a tio n of
u n c o u p le d 3* o r 5' incisions, the sin g le lesion D N A su b stra te w as labelled at
th e 3' en d w ith resp ect to the lesion (section 2.14.3) an d in cu b ated w ith cell
extracts (Fig. 5.6). This assay is d esig n e d to d etect all 3 ’ incisions, arising
d u rin g th e d u a l incision re a c tio n or as u n c o u p le d 3' o r 5' incisions (see Fig.
5.5 a n d d ia g ra m s on the rig h t of Figure 5.7). All 3' incisions are d etected , b u t
5' incisions are only d etected if m ad e in the absence of 3' incisions.
in cisio n 3 'in cisio n
K
&5 'in d s io n 3 'in c isio n
5 -G T C — ■— — — ' C A G - 5 ' 6 .9 kb 140 b p |--- 1 3' u n c o u p le d in c is io n s 1 3 1 /1 3 2 b p + d u a l in c is io n s I I 5' u n c o u p le d in c is io n s 1 5 7 /1 6 0 / 1 6 1 b p F ig . 5 .5 S c h e m e r e p r e s e n t in g t h e s i z e s o f t h e f r a g m e n t s g e n e r a t e d b y d u a l in r is io n o r u n c o u p le d i n c i s i o n s o n t h e A v aII c u t P t-G T G s u b s tr a te ; t h e s ta r r e p r e s e n t s th e s i t e o f 3 ’ l a b e l l i n g o f th e a d d u c t e d s tr a n d .
Extracts from XP8BR, XP7BE, TTD5BR a n d XP1NE XP-D cells w ere
co m p a re d w ith XP11BE, w h ich co n tain s the m u ta n t XPB h elicase th a t
displays 3' u n c o u p le d incision activ ity (Evans et al. 1997b) as well as XP20S
(XP-A cell line GM2345) an d XP2BI (XP-G cell line XPG415A) b o th k n o w n to
be com pletely defective in 3' an d 5' in cisio n fo rm a tio n (Evans et al. 1997a;
E vans et al. 1997b).
C h ap ter V - XPD m u tan ts
• + ■o >
| g a -1 — X P A | — H e p | 25 50 H e p | 25 50 H e p |2 5 50 H e p ||2 5 50 H e p ||- X P G |
HeLa XP-A XP-B XP8BR TTD5BR XP1NE XP7BE XPG >
I_________________________________ 1415A
mutated in XPD
Fig. 5.6 - In cision activity of XPD mutant cell lines.
X P -D m u t a n t W C E s w e r e te s t e d fo r th e ir 3 ' a n d 5' i n c i s i o n a c t i v it y in c o m p a r i s o n w it h c e ll e x tr a c t s w i t h k n o w n in c i s i o n p a tte r n s ; th e a m o u n t s o f e a c h W C E w e r e a s f o l l o w s H e L a - 25 p g ; X P -A ( X P 2 0 S ) - 2 5 p g ; X P -B (X P 1 1 B E ) - 2 5 p g ; X P G 4 1 5 A - 2 5 p g a n d th e v a r io u s X P - D c e ll e x tr a c t s w e r e a s in d ic a te d ; la n e s c o n t a in in g T F IIH H e p (la n e s 6, 9 , 1 2 , 1 5 a n d 18 ) h a d 2 p i; X P A - 1 0 0 n g ; X P G - 5 0 n g ; a ll l a n e s e x c e p t l a n e 1 c o n t a i n a p h i d i c o l i n to a f in a l c o n c e n t r a t io n o f 6 0 p M ; la n e 21 c o n t a in e d o n ly r a d io l a b e ll e d D N A s u b s t r a t e n o t s u b j e c te d to a n y r e a c t io n ; a r r o w s i n d i c a t e th e p o s i t i o n s o f 3' a n d 5' in c i s i o n s , fo r m o r e d e t a i l s e e f ig u r e s 5 .5 a n d 5 .7 . 147
C h ap ter V - X PD m u ta n ts M l 2 3 4 5 6 7 8 9 10 11 12 13 181 — 161 — 148 — 124 — 111 — u n co u p led
S'
in c is io n s 3' incisions-
T F I I H I T F I I H H e p X P G wt wt — E 7 9 1 A w * wt wt wt w * w * wt b> ■ hH M l-rt h—I ^ ^ ^ J RPA, XPA, XPC-hHR23B, XPG, ERCC1-XPFFig. 5.7 - Incision activity o f purified TFIIH containing mutated XPD helicase.
P u r ifie d T F IH c o n t a in in g a d e f i n e d m u t a t io n in X P D h e lic a s e s u b u n i t w a s te s t e d fo r 3' a n d 5' in c i s i o n a c tiv ity ; L a n e 1 - n o T FIIH ; la n e s 2-5 c o n t a in e d 1.5 p i o f H e p T FIIH ; la n e 3 h a d n o X P G a n d la n e s 4 a n d 5 c o n t a i n e d 0 .7 5 n g a n d 1.5 n g , r e s p e c t iv e l y , o f E 7 9 1 A m u t a n t X PG ; la n e s 6 -1 2 c o n t a i n e d t h e i n d i c a t e d T F IIH c o m p l e x e s : 1 .5 a n d 3 p i o f w t a n d K 4 8 R T FIIH , 3 p i o f T F IIH i m m u n o p u r if ie d f r o m H e L a , H D 2 a n d T T D 1 B R c e ll e x tr a c ts; la n e 13 c o n t a in e d o n ly r a d io la b e lle d D N A su b s tr a te ; p o s i t io n s o f in c is io n s a r e a s in d ic a te d .
C h a p te r V - XPD m u tan ts
R eactions (w ith the exception of lane 1) w ere p e rfo rm ed in the presence of aphidicolin (inhibitor of polym erases 5 and 8, that are im plicated in NER (Shivji et a l 1995)) and in the absence of nucleotides. This w o u ld enable the incision events to be detected avoiding further processing by the replication m achinery p resen t in these extracts. Interestingly, aphidicolin also in h ib ite d som e 5 '—>3' exonuclease a ctiv ity in th e cell extracts. C om parison of lanes 1 and 2 show s th at in the absence of aphidicolin (lane I) there w as some degradation of the labelled p roduct that cannot be detected w hen this inhibitor is ad d ed (lane 2). 3' incisions w ere form ed by repair proficient HeLa (Fig. 5.6 lanes 1-2), and a low er level by TTD5BR (lanes 10- II) and XP1NE (lanes 13-14) extracts all of w hich m ake dual incisions (Fig. 5.2).
A dditionally, 3' incisions could be detected u p o n com plem entation of any of the cell extracts w ith TFIIH Hep (Fig. 5.6 lanes 9, 12,15,18). Uncoupled 3' incisions w ere not detected except in the reactions containing XP11BE extracts (Fig. 5.6 lane 5).
N o o th er incisions w ere detected; 5' u n co u p led incisions w ere not id en tified n o r w ere any o th er specific incisions a ro u n d the lesion or anyw here else in the plasm id substrate, w ith any of the cell extracts tested (Fig. 5.6).
This stu d y was p erform ed u sing w hole cell extracts. A lthough the DNA polym erase activity h ad been inhibited, one cannot com pletely rule o u t the possibility th at other enzym e activities in the cell extracts could m ask the incisions, d eg rad e them , or obscure their detection. To further clarify the 3' an d 5' incision activity of XPD m u tan t TFIIH, purified TFIIH containing a m utant K48R XPD (section 5.3) and w t TFIIH w ere tested in the assay th at detects 3' and 5' incisions, in com bination w ith other purified NER proteins (Fig. 5.7).
In the presence of w ild type TFIIH, 3' incisions w ere readily form ed (d u rin g the d u al incision reaction - Fig. 5.2). No 5' or 3' incisions w ere
C h a p te r V - XPD m u ta n ts
d etected w h en the reactions w ere p erfo rm ed w ith TFIIH h a v in g K48R m u tan t XPD subunit (Fig. 5.7, lanes 6-9). As a positive control, uncoupled 5' incisions w ere efficiently placed in the presence of E791A m u ta n t XPG protein as previously observed (C onstantinou et al. 1999). 3' incisions w ere also form ed by TFIIH im m unopurified from HeLa, HD2 and TTD1 cells (Fig. 5.7 lanes 10-12), and these were all consistent w ith the dual incision activity p resent in these cells (Fig. 5.3). No additional 3' or 5' u n co u p led incisions w ere detected in reaction m ixtures containing XPD m u tan t TFIIH.