4.2 PRUEBAS DE USUARIO
4.2.1 EMPRESA A
In collaboration w ith M ark Berneburg and A lan R. L ehm ann (MRC- CMU, Sussex) different cell lines from patients w ith X P D m u tatio n s were analysed.
P a tie n t M u tatio n (am ino acid) a l l e l e l
a l l e l e2
%UDS R eferences
XP7BE Del (36-61)
R683W*
25-50% (A ndrew s et al. 1978; Johnson a n d S q u ir e s 1992) (A.R. L ehm ann, u n p u b lish e d )
XP8BR G675R*
F /s h ift 669
30-40% ( B r o u g h t o n et al. 1995) (A .R .Lehm ann, u n p u b lish ed )
XP12BR R511Q
R511Q
30-40% (A.R. L e h m a n n a n d B. C. B roughton, u n p u b lish ed )
TTD5BR A594P*
splice m u tatio n -in tro n 7
30% (M o tle y a n d F in la y 1989) (A.R. L ehm ann, u n p u b lish ed )
XP1NE L461V, Del (716-730)
G47R*
40% (L eh m an n a n d S tevens 1980; J o h n so n a n d S q u ire s 1992; T aylor et al. 1997)
T ab le 5.1 - M u ta tio n s in XPD gene in th e XPD an d TTD p a tie n t cell lin e s u s e d in th is stu d y . M utatio n s are sh o w n for both alleles an d the causative m u ta tio n m a rk e d b y * ; in the case of XP12BR, the p atien t is hom ozygous for the m u tatio n show n.
C h ap ter V - XPD m u tan ts
In order to study the NER activity of XP-D m u tan t cells, five WCEs of different cell lines established from patients of either XP group D, TTD or XP/C S were m ade, according to section 2.2. All cell lines show high levels of UDS (Table 5.1).
Three cell lines belong to XP group D (XP7BE, XP12BR and XP1NE) (Taylor et al. 1997), one to TTD (TTD5BR) (A. R. Lehm ann, unpublished) and the other to X P/C S (XP8BR) (B roughton et al. 1995). The m utations present in these XPD alleles have been identified (Table 5.1 and Fig. 5.1) and the causative m utation has been detected using S. pombe cells (Taylor et al.
1997) (A. R. Lehm ann, unpublished). Of these XP-D cell extracts, the only th at has been biochem ically stu d ied in som e detail is XP7BE (GM2485) (Evans et al. 1997b).
The NER activity of XPD m utants w as tested by d u al incision assay (section 2.14.1). Dual incisions were form ed by TTD5BR (Fig. 5.2A lanes 2-3 and C lanes 6-7) and XP1NE (Fig. 5.2C lanes 9-10) b u t not by XP8BR (Fig. 5.2A lanes 7-8 an d C lanes 2-4), XP7BE (Fig. 5.2A lane 16 an d B lanes 9-11) or XP12BR (Fig. 5.2B lanes 2-4). All cell extracts could be com plem ented by eith er H eLa p u rifie d TFIIH (TFIIH H ep - C h a p te r 4) or by cro ss com plem entation w ith CHO 43-3B WCE (defective in ERCC1). A lthough TTD5BR an d XP1NE exhibited some dual incision activity, they show ed fu rth er stim u latio n u p o n a d d itio n of com plem enting activity (Fig. 5.2A lanes 2-6 and Fig. 5.2C lanes 6-11).
G 47R K48R
\/
M P SG TG K T R511Q G675R R683WV
I la II III IV V VI XPD 51 88 239 468 553 613 671 A594PFig. 5.1 - M u ta tio n s in th e XPD p ro tein . D iagram of the XPD p ro te in w ith the seven helicase d o m ain s an d the causative m utatio n s p re sen t in cell lines u se d in this study.
^.napter v - ar u m u tan ts 6000000 5000000 £2 4000000 - - | 5 3000000 J 5 "5 O' M 2000000 - 1oooooo - JZZL lO 11 12 13 14 15 16 17 18 19 20 35 27'
I
- J X pg I I paCDi rO I I TFIIH U H eP pa CT> I pa ,I cn q TFIIH TFIIH q Q H ep H eo S pa pq cfi cfl CT> CD H ep g c d c d TFIIH J ^ H e p 5 5 JL I TTD5BR XP8BR 22 44 44 44 44 17 34 34 34 34 XP11BE XP7BE 44 44 44 44 44 44 44 44Fig. 5.2 - N ER activity of XPD m u ta n t cell lines.
A - D if f e r e n t a m o u n t s o f X P - D W C E s w e r e t e s t e d in d u a l i n c i s i o n a s s a y a s in d ic a t e d ; c e ll e x tr a c t s w e r e c o m p l e m e n t e d e it h e r b y a d d it io n o f 3 0 p g 4 3 -3 B W C E o r b y 1 o r 2 p i o f T F IIH H e p ; t h e a m o u n t o f H e L a W C E u s e d in l a n e 1 w a s 1 7 .5 p g ; r e s u l t s w e r e q u a n t if ie d o n a p h o s p h o r i m a g e r , w h e r e t h e d e n s i t y o f b a n d s c o r r e s p o n d i n g to th e 2 6 - 3 0 m e r p r o d u c t s w a s m e a s u r e d (u n it s a r e a r b itr a r y ). B - D if f e r e n t a m o u n t s o f X P -D W C E s w e r e t e s t e d in d u a l in c i s i o n a s s a y a s in d ic a t e d ; c e ll e x tr a c t s w e r e c o m p l e m e n t e d b y a d d it io n o f 1 o r 2 p i o f T F IIH H e p ; th e a m o u n t o f H e L a W C E u s e d in la n e 1 w a s 17 .5 p g . C - D if f e r e n t a m o u n t s o f X P - D W C E s w e r e t e s t e d in d u a l in c i s i o n a s s a y a s in d ic a t e d ; c e ll e x t r a c t s w e r e c o m p l e m e n t e d b y a d d i t i o n o f 2 p i o f T F IIH H e p ; th e a m o u n t o f H e L a W C E u s e d in l a n e 1 w a s 2 5 p g ; t h e a m o u n t o f X P - D W C E s u s e d in c o m p l e m e n t a t io n r e a c t io n s ( la n e s 5, 8 a n d 11) w a s 5 0 p g . 134
C hapter V - XPD m u tan ts B M l 2 3 4 5 6 7 8 9 10 11 12 13 14 15 35 1 2 7 ' a» X I I I TFIIH TFIIH Hep Hep I I I I I l I TFIIH TFIIH H ep Hep I II I ! 22 44 8 8 22 44 j ^ 25 50 100 25 50 ^ p g XP12BR XP7BE 1 2 3 4 5 6 7 8 9 10 11 s = * s a R 25 50 Pg 25 50 Pg 25 50 68 P g XP8BR TTD5BR XP1NE 135
C h ap ter V - XPD m u tan ts
The NER activity of these XP-D cell extracts can be correlated w ith the causative m utations in XPD represented in figure 5.1.
Both XP7BE and XP8BR h arb o u r causative m u tatio n s (R683W and G675R, respectively) in the C-term inal p a rt of XPD (Table 5.1, Fig. 5.1), a region w here a significant pro p o rtio n of XP group D causative m utations have been detected (Taylor et al. 1997). They are very UV-sensitive (Johnson an d Squires 1992; B roughton et al. 1995) an d the lack of dual incision form ation is in agreem ent w ith their NER deficient p h en o ty p e (Johnson and Squires 1992). R683W and G675R are am ino acid substitutions that p rev en t XPD in te rac tio n /stim u la tio n w ith p44 therefore interfering w ith the helicase activity of this protein (Coin et al. 1998b).
XP12BR harbours a m utation also found in the C-term inal half of XPD. The p atien t from w hom this cell line was established has been assigned to XP group D and is hom ozygous for the R511Q am ino acid substitution (A. R. Lehm ann and B.C. Broughton, personal com m unication). This am ino acid change, m ight also prevent an association w ith p44. H ow ever, since this is a new ly found m utation, no detailed m olecular analysis has yet been done.
The XPB m u tatio n in XP11BE cells is o u tsid e the DNA helicase dom ains, an d results in an altered C -term inal 42 resid u es (W eeda et al.
1990). A lth o u g h this TFIIH cannot su p p o rt the 5' NER incision, the 3' incision can still take place, as observed w hen w hole cell extracts from this patient w ere analysed for NER activity (Evans et al. 1997b). D uring this study XP11BE w as used as a control (Figs. 5.2 lanes 12-15, 5.4 lanes 2-5 and 5.5 lanes 5-6).
The m u ta tio n in TTD5BR resu lts in an am ino acid su b stitu tio n (A594P) in the C-terminal dom ain of XPD (Taylor et al. 1997). The causative m u ta tio n in XP1NE resu lts in an am ino acid su b stitu tio n w ith in the nucleotide binding motif (Walker type A) (Taylor et al. 1997). XP1NE is the only cell line tested in this p art of the study w here the causative m utation is in the N -term inal region of XPD (Table 5.1 and Fig. 5.1). Both TTD5BR and
C h ap ter V - XPD m u tan ts
XP1NE show ed low levels of dual incision form ation.
TTD5BR show ed dual incisions at a level w hich is about 30% that attain ed by cross-com plem entation of XP-D cell extracts w ith an ERCC1 defective extract (Fig. 5.2A lanes 4, 9, 15, 19). The activity show n by XP1NE cell extract was lower. Both activities could be further stim ulated by addition of purified TFIIH (Fig. 5.2C lane 8 and 11) or 43-3B WCE (Fig. 5.2A lane 4). As the causative m utation in XP1NE cells leads to an am ino acid substitution w ith in the A T P-binding m otif (G47R), it w as an ticip ated this m u tatio n w ould render the helicase inactive by preventing ATP hydrolysis (Taylor et al. 1997). A dditionally, a rad3 m utant allele bearing the equivalent m utation in S. cerevisiae displays extreme UV-sensitivity (Song et al. 1990). This led to an expectation of a com plete NER defect in this cell line th at w as not observed (Fig. 5.2C lanes 9-10). In section 5.4, a d etailed stu d y of TFIIH containing an XPD subunit m utated at a different am ino acid residue in this sam e W alker type A motif is reported and these m utations in XPD will be further discussed at the end of this chapter.
5.3 NER activity of TFIIH purified from cells w ith m u tan t XPB, XPD and p44
As p re v io u sly m entioned, d ifferen t m u ta tio n s in the tw o TFIIH helicase sub u n its, XPB and XPD, lead to a v ariety of h u m an syndrom es including XP, CS and TTD (review ed in (de Laat et al. 1999)). M oreover, it was found that the gene encoding p44 is duplicated in the chrom osome 5ql3 region. The m ore telomeric of the tw o genes (p44t) is located in a region associated w ith Spinal M uscular A trophy (SMA) w hereas the other (p44c) is m ore centrom eric. The two p44 gene pro d u cts differ by three am ino acids (Biirglen et al. 1997; Tirode et al. 1999). p44t is deleted in cells from patients suffering from the m ost sever form of SMA (type I SMA - W erdnig- H offm ann disease). As previously m entioned, the interaction betw een p44 and XPD was show n to be im portant for the helicase activity of TFIIH (Coin
C h a p te r V - XPD m u tan ts et al. 1998b). It w as therefore clearly im portant to exam ine the NER activity of TFIIH purified from cells carrying alterations in various subunits.
In order to do this, TFIIH was im m unoprecipitated using an antibody ag ain st the p44 su b u n it (Coin et al. 1999). In reco n stitu ted reactions the activity of TFIIH im m unopurified from the repair proficient cell lines HeLa a n d MRC5 (an SV 40-transform ed h u m a n fib ro b last, for exam ple see (M ackenney et al. 1997)) was com pared w ith TFIIH from an XP-B cell line, an XP-D cell line, a TTD group A cell line, an d tw o cell lines derived from patients w ith SMA.
As expected, TFIIH purified from HeLa an d MRC5 cells w as active in d u al incision (Fig. 5.3 lanes 3 and 5). Also, TFIIH from the XP-B cell line GM2252 - patient XP11BE w as defective in placing dual incisions around a DNA lesion (Fig. 5.5 lane 10). W hereas TFIIH purified from GM1855, a cell line derived from the asym ptom atic m other of p atien t XP11BE (H w ang et al.
1996), w as active in NER (Fig. 5.3 lane 9).
TFIIH w ith an XPD m u ta tio n w as p u rifie d from HD2 cells an d although the TFIIH supported a very low level of repair, it w as not zero (Fig. 5.3 lane 6). HD2 is an im m ortalised cell line derived from fusion of XP102LO XP-D cells w ith HeLa cells, w ith subsequent selection of a UV sensitive clone (Johnson et al. 1985). XP102LO cells carry a R683W m u tatio n at the C- term inal end of one allele, and an L416V substitution as w ell as a deletion of am ino acids 716-730 in the other allele (Takayama et al. 1995). The causative m utation in HD2 is assum ed to be R683W as this m u tatio n determ ines the phenotype in p atien t XP102LO (Taylor et al. 1997). The R683W am ino acid substitution is in a region of the X P D gene w here m an y XP-D causative m utations are p resen t (Taylor et al. 1997), and this region of the protein is involved in the interaction betw een XPD and p44 (Coin et al. 1998b).
^naprer v - A r u mucanrs M 1 2 3 4 5 6 7 8 9 10 35 27 TFIIH Oh V X ir> U &pa a» X <N D X u 3 O X X) e* 0) 0 o o <3 IT) LO 00 <N IT) (N <N U O
RPA, XPA, XPC-hHR23B, XPG, ERCC1-XPF
Fig. 5.3 - N ER ac tiv ity of im m u n o p re c ip ita te d T F IIH c o n ta in in g m u ta tio n s in d iffe re n t su b u n its.
T F IIH i m m u n o p r e c ip i t a t e d f r o m d if f e r e n t n o r m a l, m u t a t e d in X P D o r in X P B c e ll l i n e s w a s t e s t e d in 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 s in d ic a t e d ; th e a m o u n t s o f T F IIH u s e d w e r e T F IIH H e p - 1 .5 p i, M R C 5 T F IIH - 2 p i, T T D 1 B R , H e L a , H D 2 , R a b b o u c h , D ja k o u n e , G M 1 8 5 5 a n d G M 2 2 5 2 T F IIH - 3 p i.
C h ap ter V - XPD m u tan ts
R epair in HD2 cells as m easured by UDS an d sensitivity to UV was rep o rte d to be slightly higher than th at of the XP-D p aren tal cell line XP102LO (Johnson et al. 1985) and it seem s plausible th at some residual activity m ight be contributed by a low level of expression of norm al TFIIH in the hybrid HD2 cells.
The activities of TFIIH from cells co n tain in g only the p44c g e n e (Rabbouch, Fig. 5.3 lane 7) and a TFIIH containing b o th p44t an d p44c (Djakoune, Fig. 5.3 lane 8) w ere com pared. TFIIH activity in NER w as not affected by the absence of p44t. The helicase activity of XPD in these cell lines has been show n to be norm al (Coin et al. 1999) an d TFIIH containing only