18 m 4 h esims m z rel intensity 407 0 100 mh anal calcd for c 23 h 22 n 2 o 5 0 9chcl 3 c 55 86 h 4 49 n 5 45 found c 55 86 h 4 72 n 5 24 human recombinant top1 was purified from baculovirus as described by pourquier et al in j biol chem 274 8516 23 1999 the disclosure of which is incorporated herein by reference the 161 bp fragment from pbluescript sk u2212 phagemid dna stratagene la jolla calif was cleaved with the restriction endonuclease pvu ii and hind iii new england biolabs beverly mass in supplied ne buffer 2 10 u03bcl reactions for 1 h at 37 u00b0 c and separated by electrophoresis in a 1 agarose gel made in 1 u00d7 tbe buffer the 161 bp fragment was eluted from the gel slice centrilutor by amicon and concentrated in a centricon 50 centrifugal concentrator amicon beverly mass approximately 200 ng of the fragment was 3 u2032 end labeled at the hind iii site by fill in reaction with alpha 32 p dgtp and 0 5 mm datp dctp and dttp in react 2 buffer 50 mm tris hcl ph 8 0 100 mm mgcl 2 50 mm nacl with 0 5 units of dna polymerase i klenow fragment unincorporated 32 p.