Specific Sites ofInteraction Between Histones andDNA inChromatin (nudease/DNA-electrophoresis)

Staphylococcal nuclease digestion ofpuri- fiedchromatin fromduckreticulocytes orcalfthymusre- sultsintheproduction ofaseries ofdouble-stranded DNA fragments ofdiscrete molecular size, rangingfromabout 130to45basepairs, whichcanbedetected bypolyacryl- amidegelelectrophoresis. Similar patterns ofprotected DNA fragments areobtainedfromlimitdigests ofchro- matin"reconstituted" frompurified DNA andchromatin proteins. The results obtainedwithreconstituted ma- terial donotdependupontheorigin oftheDNA,which may bederivedfroma bacterial, viral, orhomologous source. Thespecificity oftheprotective mechanism,there- fore, resides inthestructure oftheboundhistones, and probably notinanyspecial nucleotide sequences present intheDNA.Removaloflysine-rich histones fromchro- matinbefore digestion results principally indisappearance fromthedigest ofaDNA fragmentabout130basepairs long.Ourpreliminary results suggest thatotherelements ofthedigest patterncanbeassigned uniquelytothere- maininghistone components. Theseresults indicate that thebindingofhistones toDNA inchromatininvolves a limited numberofspecific andverywelldefined contacts betweenproteinand nucleicacid,which arisefrom structural properties ofthehistones. Manystudies ofthebinding ofhistones totheDNA of chromatin havesuggested thatthehistones interact with DNA through welldefined sites ontheproteins (1). Other evidence indicates that thehistones maybeboundtoDNA in theformofspecific complexes involving morethanone histone species (2-4). Ithasbeenproposed (4)thatsuch complexes arearranged inaregular repeating sequence along theDNA,giving rise tothetertiary DNA structure present in chromatin. Evidence suggesting thathistone complexes may cover theDNA inaregular fashion isprovided bystudies of theproducts ofnuclear autodigestion (5,6).Thesestudies reveal thatautodigestion ofnuclei results inrelease ofDNA segments that aremultiples ofasubunit about 200basepairs inlength. Incubation ofnuclei withaddednuclease results in release offragments ofsimilar size (7). Inourlaboratory wehavemadeuseoftheenzyme staphylo- coccal nuclease asaprobe ofthestructure ofpurified chro- matin, andwehaveisolated those regions oftheDNA that are sufficiently tightly covered byprotein tobeprotected from digestion (8, 9).Abouthalf theDNA issusceptible todiges- tion. Therest isprotected, andisreduced todouble-stranded segments withaweight average length ofabout110to130 base pairs regardless oftheamount ofenzyme used(9).