CARACTERÍSTICAS DE HARDWARE

N uclear extracts from lym phoblastoid an d fibroblast cells w ere m ade according to M asutani and co-w orkers (M asutani et al. 1994) w ith some m odifications. Cell pellets w ere re su sp e n d e d in cold PBSA (4°C) and repelleted by centrifugation. The packed cell volum e w as estim ated before resu sp en d in g the cell pellet in four packed cell volum es (PCV) of hypotonic lysis buffer (10 mM Tris-HCl, p H 7.5, 0.5 mM EDTA, 2 mM MgCl2, 5 mM DTT, 2.5 m M PMSF, 10 p g /m l leupeptin, 10 p g /m l p ep statin , 10 p g /m l chym ostatin and 5% aprotinin (w /v )). Cells w ere incubated on ice for 20 m in u tes before breakage by 30 to 40 strokes of a Teflon h o m ogeniser (Jencons Scientific). The m ixture was p o u red into centrifuge tubes and spun at 3000 rp m (1500 x g) in a Sorvall HB4 centrifuge rotor at 4°C for 10 min. The su p ern atan t was rem oved and the pellet (containing the nuclei) w ashed twice w ith Nuclei W ash Buffer (10 mM potassium pho sp h ate p H 7.5, 2 mM DTT an d 2.5 mM PMSF), spinning again at 1500 x g for 10 m in at 4°C for harvesting. The pellet was then ressuspended in tw o packed cell volum es of Buffer 1 (20 mM Potassium phosphate p H 7.5, 1 mM EDTA, 5 mM DTT, 2.5 mM PMSF, 10 p g /m l leupeptin, 10 p g /m l pepstatin, 10 p g /m l chym ostatin and 5% aprotinin (w /v)) and the salt concentration w as gradually adjusted to 0.3 M KC1. After 30 m inutes stirring at 4°C the solution w as centrifuged at 36000 rp m (100000 x g) in a Beckman 45 Ti (or 60 Ti) ultracentrifuge rotor at 2°C for 60 m inutes. The pellet w as then discarded and Triton X-100 ad d ed to a final concentration of 0.01%. Dialysis was carried o u t in 2 litres of cold Dialysis Buffer (25 mM Hepes-KOH p H 7.9, 100 m M KC1, 12 mM MgCl2, 1

V^lldplei II - Ivlctlei ld.la d im iv ie in u u s

m M EDTA, 17% glycerol (v /v ) (Fluka) and 2 mM DTT) for approxim ately 12 hours. After dialysis, insoluble proteins w ere rem oved by centrifugation at 16000 x g for 10 m in at 4°C an d the clarified dialysate lo ad ed onto the a p p ro p riate chrom atography colum n.

2.4 Preparation of PCNA- and RPA -depleted (CFII) cell extracts

CFII cell extracts w ere p rep ared as described previously (Shivji et al.

1992). Briefly, w hole cell extract (from no less then 1010 cells), p rep ared as d escrib ed above, w as lo ad ed onto a p h o sp h o cellu lo se (W hatm an P l l ) colum n equilibrated in Buffer A (25 mM Hepes-KOH (pH 7.8), 1 mM EDTA, 0.01% NP40 (v /v ), 10% glycerol (v /v ), 1 mM DTT) containing 0.15 M KC1. After collection of flow -through (FT) fractions (at 0.15 M KC1, nam ed CFI), b o u n d protein w as eluted w ith Buffer A containing 1.0 M KC1. The peak p ro tein fractions from the 1.0 KC1 elution (nam ed CFII) w ere pooled and dialysed against Dialysis Buffer (25 m M Hepes-KOH (pH 7.9), 1 mM EDTA, 17% glycerol (v /v ), 1 mM DTT, 12 mM MgCl2) containing 0.1 M KC1. W hen necessary, pressure ultrafiltration th ro u g h Am icon u ltrafiltration cells w as em ployed to concentrate CFII to at least 5 m g /m l p rio r to dialysis. After dialysis, the CFII extract was spun at 3000 x g for 10 m in at 4°C to rem ove insoluble proteins. The supernatant w as frozen at -80°C in small aliquots (5- 20 pi). CFII from HeLa cells were prepared by M ahm ud K. K. Shivji.

2.5 Protein purification

2.5.1 Fractionation of HeLa extracts

Step 1 - Fresh cell pellets from a 40 1 culture of HeLa cells (approxim ately 2 x 1011 cells) were taken and a nuclear extract w as m ade according to section 2.3. A fter overnight dialysis against buffer A (25 mM H epes-KO H p H 7.8, 1

v^iicipici 11 - ivicueiitiia cum ivienujua

mM EDTA, 0.01% NP40 (v /v ), 10% glycerol (v /v ), 1 mM DTT) containing 150 mM KC1, the clarified dialysate (480 mg of total protein) w as loaded onto a 70 ml (dim ensions 15 x 2.5 cm; flow rate 2 m l/m in ) phosphocellulose colum n (W hatm an P l l ) p re-equilibrated in b u ffer A / 150 mM KC1. The colum n w as w ashed w ith the same buffer A containing 150 m M KC1 and proteins eluted w ith increasing KC1 concentrations in a stepw ise m anner (300 mM, 600 mM and 1 M). All fractions w ere dialysed against buffer A containing 100 mM KC1.

Step 2 - Protein eluting from phosphocellulose at 600 mM KC1 w as loaded onto a 5 ml (7 x 1 cm; 0.5 m l/m in ) Q -sepharose colum n pre-equilibrated in buffer A /lOO m M KC1. The colum n w as w a sh e d w ith the sam e buffer containing 100 mM KC1 and elution perform ed sequentially w ith buffer A containing 200 mM and 500 mM KC1.

Step 3 - The flow-through of Q-sepharose colum n (100 mM) w as m ade up to 100 m M phosphate and loaded onto a 10 ml hydroxyapatite (15 x 1 cm; 0.5 m l/m in ) colum n previously equilibrated in buffer B (25 m M H epes, p H 7.8, 100 mM KC1, 10% glycerol (v /v ), 0.01% NP40 (v /v ), 1 mM DTT) containing 100 mM p hosphate. After the colum n w as w ash ed , p ro tein s w ere eluted seq u en tially w ith buffer B containing 200 mM , 300 m M an d 500 mM phosphate. All fractions w ere dialysed against buffer A containing 100 mM KC1 and stored at - 80°C for further use.

2.5.2 Purification of IF7

Step 1 - N uclear extracts w ere p rep ared from approxim ately 2 x 1011 cells according to section 2.3. This nuclear extract w as dialysed overnight against buffer A (25 mM Hepes-KOH pH 7.8, 1 mM EDTA, 0.01% NP40 (v /v ), 10% glycerol (v /v ), 1 mM DTT) containing 150 m M KC1 a n d clarified by centrifugation (3000g, 10 m in, 4°C). A bout 2.5 g of n uclear extract (at a concentration of 3.5 m g /m l) w ere ap p lied onto a 300 m l (15 x 5 cm; 2

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m l/m in ) phosphocellulose colum n (W hatm an P l l ) prev io u sly equilibrated in buffer A containing 150 m M KC1. After w ash in g extensively w ith the same buffer, the elution is perform ed sequentially w ith buffer A containing 400 mM, 650 mM and 1 M KC1. Protein eluting at 650 mM KC1 (255 mg) contained the bulk of IF7 activity and w as dialysed against buffer B (25 mM Hepes-K O H p H 7.8, 10% glycerol (v /v ), 0.01% NP40 (v /v ), 1 mM DTT) containing 100 mM phosphate.

Step 2 - After dialysis, the phosphocellulose 650 m M KC1 fraction was loaded