Polyamine-induced hydrolysis of apurinic sites in DNA and nucleosomes

Volume 10 Number 201982 N u c l e i c A c i d s Research Polyamine-induced hydrolysis of apurinic sites in DNA and nudeosomes Rune Male, Viggo M.Fosse and Kjell Kleppe Department of Biochemistry, University of Bergen, Bergen, Norway Received 14 September 1982 ABSTRACT T h e a b i l i t y o f d i f f e r e n t p o l y a m i n e s to c a t a l y z e h y d r o l y s i s of p h o s p h o d i e s t e r l i n k a g e s in apurinic and a p y r i m i d i n i c (AP) sites h a s b e e n i n v e s t i g a t e d in s u p e r c o i l e d , relaxed and d e n a t u r e d D N A , a n d a l s o in core and c h r o m a t o s o m e p a r t i c l e s . The rate c o n s t a n t s for the hydrolysis in t h e DNAs have b e e n d e t e r m i n e d . In g e n e r a l t h e o r d e r of e f f e c t i v e n e s s o f the polyamines were: spermine >spermidine >putrescine >cadaverine. A 9 fold d i f f e r e n c e in rate constants w a s found b e t w e e n spermine and c a d a v e r i n e . N o d i f f e r e n c e in t h e rate o f h y d r o lysis w a s seen b e t w e e n A P - s i t e s in s u p e r c o i l e d a n d relaxed D N A s , w h e r e a s t h e rate for the s i n g l e - s t r a n d e d D N A and D N A in core and c h r o m a t o s o m e p a r t i c l e s w a s only half o f t h a t in the d o u b l e - s t r a n d e d D N A . A l l A P - s i t e s in b o t h free DNA and D N A - h i s t o n e p a r t i c l e s w e r e hydrolyzed in the p r e s e n c e o f p o l y a m i n e s . F o r all p o l y a m i n e s , w i t h the e x c e p t i o n o f s p e r m i n e , i n c r e a s i n g c o n c e n t r a t i o n of both Mg^" + and salts such a s K C 1 b o t h led to a large d e c r e a s e in t h e rate of p o l y a m i n e - i n d u c e d h y d r o l y s i s of A P - s i t e s . T h e rate o f h y d r o lysis i n c r e a s e d m a r k e l d y w i t h increasing pH in the p H r a n g e pH 6 - pH 1 1 . INTRODUCTION A p u r i n i c a n d a p y r i m i d i n i c sites in D N A (AP-sites) are formed b y s p o n t a n e o u s h y d r o l y s i s of t h e b a s e - s u g a r linkages in D N A , by severe U V - and Y-irradiation and by t h e a c t i o n o f certain D N A r e p a i r e n z y m e s , t h e DNA g l y c o s y l a s e s . A P - s i t e s can also b e g e n e r a t e d in v i t r o by exposing the D N A to acidic c o n d i t i o n s . T h e m e c h a n i s m of repair o f A P - s i t e s investigated has been in a n u m b e r o f l a b o r a t o r i e s . I t a p p e a r s repair takes p l a c e v i a t h e excision r e p a i r p a t h w a y that (1,2) . The p h o s p h o d i e s t e r l i n k a g e in the A P - s i t e m u s t f i r s t be b r o k e n and t h e s u g a r - p h o s p h a t e residue removed by n u c l e a s e s before g a p - f i l l i n g b y D N A p o l y m e r a s e and l i g a s e joining can take p l a c e . H y d r o l y s i s o f the p h o s p h o d i e s t e r IS) I RL Press Limited, Oxford, England. 0305-1048/82/1020-63058 2.00/0 l i n k a g e s in A P 6305 Nucleic Acids Research sites can be accomplished by means of specific AP-endonucleases present in the cell (3). The hydrolysis of such linkages can, however, also be achieved by a non-enzymatic mechanism in the presence of certain basic amino acids, peptides, proteins and amines(4,5). In the present report we have examined in more detail the properties of the various polyamines with regard to hydrolysis of phosphodiester linkages in AP-sites in different DNAs and also in core and chromatosome particles. MATERIALS AND METHODS Chemicals. Methyl methane sulphonate (MMS) was obtained from Merck. Dimethyl sulphate (DMS) was from Koch-Light laboratories, and [methyl- H]-thymidine (52 Ci/mmole) and 32 y- P^ATP (3000 Ci/mmole) from The Radiochemical Centre, Amersham. The nitrocellulose membrane filters (Metrical GN-6, diameter 25 mm), were from Gelman. E.coli exonuclease III was obtained from New England Biolabs. Micrococcal nuclease was purchased from Boehringer Mannheim. Spermine tetrahydrochloride, spermidine trihydrochloride, putrescine dihydrochloride and cadaverine dihydrochloride were products of Sigma Chemical Company. Enzymes. An endonuclease specific for AP-sites in DNA was isolated from mouse plasmacytoma cells (MPC 11) according to Nes (9). The enzyme recovered from the phosphocellulose column was used. The AP-endonuclease had no detectable 6306 The polyamines spermine, spermidine, putrescine and cadaverine constitute a special class of biological important amines widely distributed in nature and found mostly associated with the nucleic acids and other negatively charged macromolecules. Only limited information is available with regard to their role in phosphodiester bond hydrolysis during DNA repair (6)_. The fact that polyamines and other amines as well are capable of hydrolysing phosphodiester linkages in AP-sites was first described by Chargaff and co-workers (7,8)_. Lindahl and Andersson (4) have shown that putrescine catalyze hydrolysis of phosphodiester linkages in AP-sites in double-stranded DNA. Nucleic Acids Research aspecific endonuclease or exonuclease activity. T^-polynucleotide kinase was purified as previously described (10) and was a gift from J.R. Lillehaug. Preparation of DNAs. 0X174 RFI and RFII DNA were labelled with[methyl- H]-thymidine and isolated according to a method of Nes and Nissen-Meyer (11) . The RFI DNA contained maximum 5 % RFII DNA, and the specific activity was (0.6-1) x 10 5 cpm/pg. RFII DNA had a specific activity of 0.4 x 10 5 cpm/yg. The 0X174 [3H]-DNAs were stored in 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 10 % ethanol at 4 °C. Relaxed DNA by use of an extract from L-cells containing topoisomerase I (12).. Denaturation of 0X174 RFII was achieved by incubation of the DNA at 100 °C for 5 min. After heating the solution was quickly frozen in ethanol-dry ice followed by slow thawing on ice. Preparation of [ 3H] -labelled and 5'-[ P]-labelled core particles and chromatosomes. [ H] -labelled core particles were prepared from L-cells by growing the cells in the presence of 1 pM [methyl- H] -thymidine (50 yCi/ymole) for 23 generations (13), and the further purification were essentially as previously described (14) , except that the reaction mixture was made 0.1 M KC1 after the micrococcal nuclease treatment. The core particles were further purified using sucrose gradient centrifugation (5-28.8 % sucrose'in 1 mM EDTA, 36.000 rpm for 18 hrs in a Beckman L8-70 centrifuge, SW 41Ti rotor).. The pellet from the 0.1 M KC1 centrifugation, containing chromatosomes and compact dimers, was resuspended in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and 0.1 mM PMSF. Insoluble material was removed by centrifugation. The chromatosomes and compact dimers were purified by sucrose density gradient centrifugation as described above. The purified DNA from core particles and chromatosomes had a specific activity of 800-2600 cpm/Ug DNA. In some experiments the 5'-OH ends of unla (...truncated)