3.3 PÁGINA WEB
3.3.2 DISEÑO
3.3.2.1 Instalación y configuración de Django
3.3.2.1.1 Proyecto
N ucleotide excision repair synthesis w as previously reconstituted in
vitro u sin g m ultiple lesion U V -dam aged p lasm id DNA substrates and a
m ixture of proteins purified from HeLa cells, bacteria, and insect cells, and DNA polym erase com ponents purified from calf thym us (A boussekhra et ah, 1995; Shivji et ah, 1995). It was found that calf thym us DNA polym erase 8 could fu n ctio n in rep a ir in conjunction w ith the recognition-incision p ro tein s (A boussekhra et ah, 1995). The availability of the highly purified recom binant incision system described above allow ed us to determ ine if these p roteins could be com bined w ith DNA polym erase com ponents in order to obtain full repair.
n h3 n h3 \ w /
\A
K 1 y I =GTCGACCAGGCCTCTTCnCTGTGCACTCTTCTTCTCCCCAGGI 1 5 ’ 1 2 7 33 38 57 A B C D E M 1 2 3 4 5 6 7 E 2 7 — 0 } 2 I SC I I B S TFIIH g « rt> n > ^ ^ +n
gNH U l II 1—L.-l. I 3 exo-free 6 8 8 £ Z p o llFig. 4.10 - R econstitution o f repair syn th esis w ith recom binant and hum an purified factors.
A - Schem atic re p re s e n ta tio n of the D N A su b stra te co n tain in g a sin g le 1,3-in tra stra n d d(G pT pG ) cisplatin crosslink w ith the sizes of the frag m en ts g en e rated b y BsfNI restriction d igestion show n u n d ern eath ; the arro w s indicate m a p p e d positions of incisions (Moggs, et al
1998)
B - R econstitution of re p a ir synthesis on a single lesion plasm id; d u al incision reactions w ere perfo rm ed by RPA, XPA, XPC-hHR23B, XPG, ERCC1-XPF an d TFIIH H ep w h e re indicated or by RPA an d CFII; after incisions, synthesis w as p erform ed by ad d itio n of PC N A (lanel), exo- free pol I (lanes 2 an d 3) an d PCNA, polym erase 8 or e (as indicated), RFC an d DN A ligase I.
C h ap ter IV - R econstitution of NER
Instead of using the U V -irradiated DNA described previously, I used the DNA containing a single specifically located cisplatin lesion, Pt(GTG). For DNA polym erase com ponents only highly p u rified h u m an enzym es instead of com ponents from calf-thym us w ere used. Finally, system s were checked using both DNA polymerase 8 and e, as gap-filling studies suggested that either polym erase could w ork for synthesising DNA in NER generated incision gaps (Shivji et al., 1995; W ood and Shivji, 1997).
The rep a ir synthesis assay u sed allow s follow ing specific repair synthesis of a defined adduct. For this p urpose, the sam e dam aged DNA molecule was used as in the incision assays p resen ted above, b u t reaction m ixtures in clu d ed a [32P ]-deoxynucleotide so th a t p atch es w o u ld be radiolabelled d uring repair synthesis. Cleavage of the closed circular M13 m olecule containing the cisplatin ad d u ct w ith B s t N l restrictio n enzym e generates a 33 nucleotide fragm ent that includes the repair site, and several larger fragm ents (Moggs et al., 1996). Synthesis arising specifically from filling the 24-32 nt gap during NER should be largely confined to the 33 nt fragm ent (labelled "C" in Fig. 4.10A), w ith some specific synthesis in the 68 nt fragm ent (labelled "B" in Fig. 4.10A) because som e m ap p ed 5' incision sites fall w ithin this fragm ent (Fig. 4.10A).
First, reconstitution experiments were carried out in tw o stages (i) dual incision/excision and (ii) repair synthesis. The reaction m ixtures included recom binant RPA, XPA, XPC-hHR23B, XPG, ERCC1-XPF, and TFIIH H ep to carry out the dual incisions. R ecom binant PCNA (Shivji et a l, 1992) and ligase I (M ackenney et a l, 1997), in addition to purified h u m an RFC and polym erase 8 or e (Fig. 4.10 lanes 4-7)) or exo-free pol I (Fig. 4.10 lanes 2 and 3) w ere used to perform the synthesis step. As a positive control, dual incision reactions w ere also accom plished by recom binant RPA and the crude fraction CFII (HeLa w hole cell ex tract after frac tio n a tio n on phosphocellulose to retain RPA and PCNA (Shivji et a l, 1992; W old and Kelly, 1988)) (Fig. 4.10 lane 1) and the synthesis step accom plished by
C h ap ter IV - R econstitution of NER
addition of PCNA. CFII fraction contains all the factors necessary for repair synthesis except RPA and PCNA. A dding RPA to a CFII fraction will allow this reaction to perform dual incision and by further addition of PCNA this m ixture w ill be able to fill in the gap generated by dual incision/excision (Shivji et ah, 1992).
Both pol 5 and pol e could function in repair. The levels of synthesis observed w ith the two hum an polym erases w ere higher than w ith exo-free pol I an d w ere specifically d ep en d en t on the ad d itio n of TFIIH (Fig. 4.10 lanes 4-7).
In o rd er to analyse only the synthesis step, repair interm ediates w ere prepared. Single lesion substrate DNA, Pt(GTG), w as incubated w ith RPA and CFII fraction in order to allow the incisions to occur. In the absence of PCNA, incision and excision of a dam aged oligonucleotide can occur, b u t rep air sy n th esis by the PCNA d e p e n d e n t D N A rep a ir p o ly m erase is p rev en ted (Shivji et ah, 1992; Shivji et ah, 1995). A fter incisions, g ap p ed DNA m olecules w ere purified by phenol-chloroform extraction and ethanol precipitation and then used as substrate for gap-filling by DNA polym erase 5 or e (Fig 4.11). D N A p o ly m erase e and 5 as w ell as exo-free pol I, CFII+RPA+PCNA and a XPA defective cell extract (XP20S) can specifically synthesise D N A on the g ap p ed rep a ir in te rm e d iate (Fig. 4.11). DNA polym erase e is dependent on PCNA and RFC as can be observed by the lack of repair p ro d u cts w hen RFC and PCNA are absent (Fig. 4.11 lanes 7, 8). H um an p u rified RFC has an activity com parable to calf-thym us purified RFC (cRFC - Fig. 4.11 lanes 3-6). H um an pol 5 has an activity comparable to calf thym us pol 5 (Fig. 4.11 lanes 10, 11) and to hum an pol s (Fig. 4.11 lanes 5 and 11). These results dem onstrate th at h u m an polym erases 8 and e and acessory factors can fill in the gap generated by dual incision/excision of a DNA lesion in vitro.
M l 2 3 4 5 6 7 8 9 10 11 12 D 27 ^ 5 ^ ^ ^
3 n n n n
n z + ^ ^
% 3 0.2 0.4 0.8 U > X p ° +n
z
>
a c
x *aPolym erase 8 Pol 8
Fig. 4.11- R econstitution of repair syn th esis w ith recom binant and hum an purified factors.
R econstitution of re p air sy nthesis on a single lesion plasm id ; d u al incision reactio n s w ere p erfo rm ed by RPA an d CFII; after incisions, D N A w as p u rifie d and synthesis p erfo rm ed by a d d itio n of R PA +PC N A +CFII (lane 1), exo-free po l I (lane 2), p o ly m erase 8 (lanes 3-9), p o ly m erase e (lanes 10 a n d 11) or XP-A extract; a d d itio n or om ission of o th er factors is as indicated; cRFC - calf th y m u s RFC; RFC - h u m a n purified RFC; Pol 8 "c" - calf th y m u s Pol 8; Pol 8 "h " - h u m a n purified pol 8.
C h a p te r IV - R econstitution of NER
Results present so far indicate that repair synthesis by purified hum an DNA polym erases can take place on a repair interm ediate th at is the result of dual incision and DNA purification (Fig. 4.11) and additionally, hum an p u rified polym erases can act sim ultaneously to the incision step w hen TFIIH H ep is used (Fig. 4.10). In the next step repair synthesis w as tested in reactions w here the incisions w ere being perform ed by recom binant factors. R econstitution w as carried out w ith all the proteins required for NER in the sam e reaction m ixture (12.5 pi volume) and results are p resented in figure 4.12.
Activity w as observed w ith TFIIH purified from HeLa cells (Fig. 4.12 lanes 2, 3 and 9, 10) as well as w ith 6-subunit recom binant TFIIH (lanes 6, 13) an d 9-subunit recom binant TFIIH (lanes 7, 14). N o rep air w as observed w hen TFIIH was om itted (Fig. 4.11 lanes 1 an d 8). U nder these conditions (approx. 70 mM salt) both the pol 8 and pol s reactions w ere d ep en d en t on PCNA (Fig. 4.11 lanes 5, 12) and the A TP-dependent PCNA loading factor RFC (Fig. 4.11 lanes 4 and 11). Both purified h u m an DNA polym erases were able to fill in the gap produced by only recom binant incision proteins.
Taken to g eth er these results show th at rep a ir sy nthesis by DNA polym erase 8 or s can take place in a DNA m olecule w here the incisions have been placed by a full set of recom binant factors.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 124 — 111 — 91 — 77 — 68 — D 35 27 t f i i h I f f i p c s f f i a a i s f f i K f f i a a I rc w a> w 2 2 1 ft w w w 2 2 - T 3 - T 3 - T 3 - T 3 5 C D C ^ ^ O' ^ ^ \D S 3 S 3
n z
n z
w > w >Polym erase 8 Polym erase £
Fig. 4.12 - R e c o n s titu tio n of re p a ir s y n th e s is w ith re c o m b in a n t a n d h u m a n p u rifie d factors. R e c o n s t i t u t i o n o f r e p a ir s y n t h e s i s o n a s i n g l e l e s i o n p la s m id ; d if f e r e n t T F IIH p r e p a r a t io n s w e r e u s e d in r e a c t io n s p e r f o r m e d in th e p r e s e n c e o f p o ly m e r a s e 8 ( la n e s 1 -7 ) o r e ( la n e s 8 -1 4 ); la n e s 2 a n d 9 c o n t a in 1 .5 p i T F IIH H e p fr. IV , la n e s 3 -5 a n d 1 0 -1 2 c o n t a in 3 p i T F IIH H a p fr. V I, la n e s 6 a n d 13 c o n t a in 3 p i rIIH 6 a n d la n e s 7 a n d 14 c o n t a in 3 p i rIIH 9; r e a c t io n s in la n e s 3 a n d 11 a r e d o n e in th e a b s e n c e o f R FC a n d r e a c tio n s in la n e s 5 a n d 12 a re d o n e in th e a b s e n c e o f P C N A .
A
C h a p te r IV - R econstitution of NER