Zona desmilitarizada DMZ

XPA binds dam aged DNA and plays a central role in NER interacting w ith m any core rep air factors (review ed in (W ood 1999) and chapter I). H aving analysed the complexes im m unoprecipitated from lym phoblastoid cells I set out to study the influence of XPA protein in the form ation of these complexes. Extracts w ere p rep ared from a lym phoblastoid cell lacking XPA protein. GM2345 is a lym phoblastoid cell line derived from patient XP20S. This cell line is com pletely defective in NER, show ing low levels of reduced size XPA m RNA (Satokata et al. 1990) and no detectable XPA protein ((M iura et al. 1991) and D. Batty, u n p u b lish ed observations). TFIIH was im m u n o p re c ip ita te d fro m GM2345 ly m p h o b la sto id cell e x tra ct as previously. A lthough this complex w as isolated from XPA deficient cells, its activity in NER was similar to the activity of an HeLa purified TFIIH (Fig. 6.9 lanes 2-4) an d co m p arab le to a TFIIH im m u n o p u rifie d from norm al lym phoblastoid cells (Fig. 6.9 lanes 3-4). H ow ever w hen the complexes co- im m u noprecipitated w ith this factor w ere further analysed, the difference d u e to the lack of XPA protein in this extract could be detected. As w ith im m u n o p re c ip ita te s from n o rm al ly m p h o b la sto id cells an in teractio n betw een TFIIH and XPC-hHR23B, XPG and a very small quantity of ERCC1-

C h ap ter VI - NER com plexes

XPF complex w as detected (Fig. 6.9 lanes 7, 8 and 9). H ow ever, as expected, no XPA protein w as present in this im m unoprecipitate (Fig. 6.9 lane 6).

To d ire c tly d e te rm in e w h e th e r th e XPA level in flu e n c e d the in te ra c tio n s b e tw e e n TFIIH a n d th e o th e r in c is io n fa c to rs I im m u noprecipitated TFIIH from XP-A cells th at h ad been supplem ented w ith pure recom binant XPA protein (to the same ratio protein/cell-extract as in com plem entation for NER activity (Koberle et al. 1999)). XPA protein was ad d ed to a GM2345 w hole cell extract and incubated for 30 m in at 30°C. Follow ing this incubation, im m u n o p recip itatio n s w ere p erfo rm ed w ith anti-cdk7 antibody and beads w ashed at 50 mM KC1, 0.01% Triton X-100. Some of the p rotein-protein interactions observed this w ay are increased com pared to the ones in the absence of XPA protein. First, I observed co- im m u n o p recip itatio n of XPA w ith TFIIH m eaning th at the recom binant XPA protein added as a com plem enting factor is now interacting w ith TFIIH complex. Second, XPC complex and XPG were detected as previously, but the am ounts of ERCC1-XPF complex w ere slightly increased. C om parison of the com plexes isolated before and after XPA com plem entation suggests that ad d itio n of XPA increases the interaction b etw een the TFIIH, XPA and ERCC1-XPF. A dditionally it show s that recom binant XPA can interact w ith TFIIH com plex w h en a d d ed to a ly m p h o b lasto id cell extract w ith no detectable levels of XPA protein and so in order for complexes to be detected by this technique, they do not necessarily have to be pre-assem bled in cells.

C h ap ter VI - NER com plexes 120% 120% 100% </> % I 80% '.fl v £ SP S E B '9 o o m J3 60% 2 1 -4 0 % ^ o "20% ■ 0% -100% - 80% J_5rT 60% I 2 40% 20% 0% 10 11 12 13 14 15 16 35 27

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H ep im m unoprecipitated TFIIH im m unoprecipitated TFIIH

TFIIH from XP-A from XP-A + XPA

Fig. 6.9 - P ro te in -p ro te in in te ra c tio n s in XP-A a n d XPA c o m p le m e n te d XP-A cell extracts. C d k 7 b e a d s w e r e u s e d in d u a l i n c i s i o n a s s a y a fte r in c u b a t io n w i t h a n X P -A W C E . 3 p i m a g n e t ic b e a d s c o n t a i n in g T F IIH a n d a s s o c ia t e d f a c t o r s w e r e a d d e d to a r e c o n s t it u t e d d u a l in c i s i o n a s s a y a n d p r o t e i n - p r o t e i n in t e r a c t io n s te s t e d b y s e q u e n t ia l o m i s s i o n o f e a c h r e p a ir fa c to r a s in d i c a t e d . L a n e s m a r k e d " C o m p le te " c o n t a i n a ll t h e r e c o m b i n a n t r e p a ir f a c t o r s a n d i m m u n o p r e c i p i t a t e d T F IIH . L a n e s 3 -9 c o n t a i n r e a c t i o n s w i t h T F I I H im m u n o p r e c ip i t a t e d f r o m a n X P -A c e ll lin e ; la n e s 1 0 -1 6 c o n t a i n r e a c t i o n s w i t h T F I IH i m m u n o p r e c ip i t a t e d f r o m a n X P - A c e ll li n e a fte r a d d it io n o f p u r if ie d X P A p r o t e in . L a n e 2 c o n t a i n s 1.5 p i H e p T FIIH ; la n e s 3, 4 a n d 10, 11 ( C o m p le t e (1 ) a n d (2 )) c o n t a in 1.5 p i a n d 3 p i o f c d k 7 m a g n e t i c b e a d s , r e s p e c t i v e l y ; l a n e s 5 -9 a n d 1 2 -1 6 c o n t a i n 3 p i o f b e a d s ; q u a n t if ic a t io n w a s p e r f o r m e d o n a p h o s p h o r i m a g e r r e la t iv e l y t o th e la n e " c o m p le t e " th a t c o n t a in s 3 p i IP T F IIH b e a d s ( la n e 4 to th e fir st s e t o f v a l u e s a n d la n e 11 fo r th e s e c o n d se t). 178

C h a p te r VI - NER com plexes

6.7 D iscussion

For the first time, I used an approach that allows the isolation of higher order complexes from m am m alian cells followed by testing for their activity in an assay th at detects the dual incisions characteristic of NER. Using this assay I w as able to test for functional interactions betw een NER factors involved in the first steps of the reaction.

Im m u n o p recip itatio n of TFIIH from HeLa cells w ith an anti-cdk7 an tibody yielded TFIIH com plex-bound factors involved in d u al incision fo rm atio n . M ost in terestin g ly , this im m u n o p rec ip ita tio n succeeded in isolating approxim ately 100% of all the TFIIH complexes in the HeLa whole cell extract. This observation indicates that m ost of the TFIIH present in the cell extract is actually complexed w ith the CAK heterotrim er, in a 9 subunit com plex, in contrast w ith w h at w as indicated by previous studies w here d istin c t com plexes (TFIIH, XPD-CAK an d CAK) hav e been rep o rte d (A dam czew ski et al. 1996; D rapkin et al. 1996; Yankulov an d Bentley 1997). These p artial TFIIH complexes are probably generated by the purification process itself. U n d er hig h salt concentration TFIIH can dissociate into several subcom plexes: CAK, CAK-XPD, core TFIIH an d core TFIIH-XPD (Schaeffer et al. 1994; Rossignol et al. 1997). A pparently, 9 su b u n it TFIIH dissociates into several forms du rin g the various colum n purification steps. A ccording to the stu d ies p resen ted in this thesis, u sing m ild extraction co n d itio n s, m o st TFIIH is p re se n t in cells as a 9 su b u n it com plex. A d ditionally, this TFIIH complex is active in NER alth o u g h it has been isolated via its interaction w ith CAK. C om plexes im m u n o p recip itated in the sam e w ay have been show n to be also active in transcription (Ossipow et al. 1995). This differs from observations in Saccharomyces cerevisiae w here a n in e-su b u n it TFIIH is required for transcription (as in h u m an cells) b u t a six-subunit core-TFIIH devoid of kinase com ponents is m ainly associated w ith rep a ir p ro tein s to form the repairosom e th a t p articip ates in NER

C h a p te r VI - NER com plexes

(Svejstrup et al. 1995). In this stu d y , I could n o t detect any significant am ount of 6 subunit TFIIH in w hole cell extracts (Fig. 6.1).

The results p resen ted in this chapter suggest th a t a fraction of the proteins required for the dual incision step of NER functionally interact in vitro u n d er m ild salt concentrations. This fraction is very significant w hen XPC and XPG are concerned. A bout 36% of all the XPC and 15% of all the XPG present in a HeLa whole cell extract is complexed w ith TFIIH at 50 mM KC1. For other NER factors, only fractions sm aller than 10% are detected in the TFIIH b o u n d fraction. H ow ever, w hen the TFIIH b o u n d fraction is supplem ented w ith recom binant RPA and used in a dual incision assay, its activity in this assay is similar to the activity of the sam e am ounts of protein ad d ed together to perform the sam e reaction. Thus a functional complex can be form ed at 50 mM KC1.

If the salt concentration is increased to an ionic strength closer to the p h y sio lo g ical level, like 150 m M KC1, the only in teractio n s th at are functionally detectable are betw een TFIIH and XPC-hHR23B and TFIIH and XPG (Fig. 6.6). A dditionally, complexes im m unoprecipitated from a cell line th at does n o t contain detectable levels of XPA protein, show presence of only XPC-hHR23B and XPG and low levels of ERCC1-XPF (Fig. 6.9). U pon a d d itio n of XPA protein, how ever, TFIIH is im m unoprecipitated n o t only w ith XPC-hHR23B and XPG b u t also w ith XPA and slightly higher levels of ERCC1-XPF.

Taken to g eth er these results indicate th at the strongest interactions am ongst NER factors are betw een TFIIH and XPC-hHR23B and TFIIH and XPG, w ith the TFIIH-XPC complex interaction being the strongest.

A schem atic d raw in g of the interactions b etw een TFIIH an d other in cisio n /ex cisio n factors, is in figure 6.10 w here the stronger and w eaker interactions have been colour coded. The strongest interaction is betw een XPC-hHR23B an d TFIIH, follow ed by the interaction of XPG w ith TFIIH complex.

C hapter VI - NER com plexes RPA TFIIH complex XPC- HHR23B XPF ERCC1 XPA w eaker stronger

Fig. 6.10 - S chem atic re p re se n ta tio n of p ro te in -p ro te in in te ra c tio n s of h u m a n NER factors.

M o d e l o f th e in t e r a c t io n s s t u d ie d in h u m a n c e ll e x tr a c ts; d a r k e r t o n e r e p r e s e n t s s t r o n g e r in t e r a c t io n a n d lig h t e r to n e , w e a k e r in t e r a c tio n a s d e t e c t e d b y fu n c t io n a l a s s a y s .

C h ap ter VI - NER com plexes

A complex of TFIIH-XPC-hHR23B-XPG can be detected both at 50 mM and at 150 m M KC1 (Fig. 6.5 and 6.6). Based on evidence com ing from m obility shift assays it was proposed that TFIIH interacts w ith either XPC or XPG an d never w ith both (W akasugi and Sancar 1998). A ssum ing th at XPC and XPG do not interact directly, the data presented here are not consistent w ith this m odel since TFIIH can be com plexed w ith both XPC an d XPG in cell extracts (Figs. 6.5 and 6.6). The assum ption th at XPC-hHR23B and XPG do not directly interact is m ost probably correct. First, it is know n th at XPG interacts directly w ith TFIIH subunits (Iyer et al. 1996). Second, from this study w e know that XPC-hHR23B is found in a complex w ith TFIIH in the absence of XPG (Fig. 6.6). The m ost direct w ay of testing for this direct interaction betw een XPC-hHR23B and XPG w ould be by doing the sam e XPG im m unoprecipitation from TFIIHA cells, w hich is impossible.

O n the level of the w eaker interactions, we have XPA and ERCC1-XPF. XPA is well know n to interact w ith ERCC1 (Li et al. 1994; Park and Sancar 1994; Li et a l 1995b; Saijo et al. 1996; Bessho et al. 1997; He an d Ingles 1997) and w ith TFIIH complex (Park et al. 1995; N ocentini et a l 1997). Also, w hen attem pts w ere m ade to isolate higher order complexes from XPA defective cells w e could detect hardly any am ounts of ERCC1-XPF in the absence of XPA protein, w hereas w hen this factor was ad d ed to the sam e extract the am ounts of ERCC1-XPF complex increased sim ultaneously. Based on all these data, and also on the fact that ERCC1-XPF w as never found to interact w ith TFIIH in m am m alian cells, these w eaker interactions w ere represented as XPF-ERCC1-XPA-TFIIH w here TFIIH complex does not contact ERCC1- XPF. RPA is know n to interact w ith XPA (He et a l 1995; Lee et a l 1995; Li et a l 1995a; M atsuda et a l 1995; Saijo et a l 1996; Stigger et a l 1998), how ever two of its subunits w ere not detected by im m unoblot in the TFIIH bound fraction. A dditionally TFIIH bound fractions at 50 mM KC1, w ere only active upo n the ad d itio n of heterotrim eric recom binant RPA. C onsequently, RPA is rep re se n te d o utside this "interactions com plex", since no physical or

C h a p te r VI - N ER com plexes

functional association w as detected. The XPA-RPA in teractio n dom ains have been stu d ied and it is thought th at this interaction is very im portant for NER (Li et al. 1995a). Perhaps the RPA-XPA interaction is only strongly form ed in the presence of DNA (W akasugi and Sancar 1998; W akasugi and Sancar 1999) or only XPA protein that is not interacting w ith TFIIH is able to interact w ith RPA. M atsuda et al. w ere able to im m unoprecipitate RPA and XPA from HeLa cell extracts using an antibody against XPA (M atsuda et al.