PNAS-1994-Panyutin-2021-5_001

PNAS-1994-Panyutin-2021-5_001 - Proc.Nat!.Acad. Sci. USA

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Unformatted text preview: Proc.Nat!.Acad. Sci. USA Vol.91,pp.2021-2025,March1994 Biochemistry The kineticsofspontaneous DNA branchmigration IGOR G. PANYUTIN AND PEGGY HSIEH GeneticsandBiochemistryBranch,NationalInstituteofDiabetesandKidneyandDigestiveDiseases,NationalInstitutesofHealth,Bethesda, MD 20892 Communicated byHowardA. Nash,November 1,1993 ABSTRACT Animportantstepingeneticrecombinationis DNA branchmigration,themovementoftheHolliday junction orexchangepointbetweentwohomologousduplexDNAs. We have determined kinetic parameters ofspontaneous branch migrationasafunctionoftemperature and ionicconditions. The branch migrationsubstratesconsistoftwo homologous duplexDNAseachhavingtwosingle-strandtailsatoneendthat arecomplementarytothecorresponding singe-strand tailsof the other duplex. Upon rapid annealing ofthe two duplex DNAs, a four-stranded intermediate is formed that has a Hollidayjunctionatoneendoftheduplexes.Branchmigration totheoppositeendoftheduplexesresultsin completestrand exchangeand formationoftwoduplexproducts. The rateof branchmigrationisexceedinglysensitivetothetypeofmetal ionspresent. In magnesium, branch migration isquiteslow withasteptime, T,equalto300msec at37C. Surprisingly, branchmigrationintheabsenceofmagnesiumwas1000times faster. Despite this difference in rates, apparent activation energiesfor the branch migrationstep inthe presence and absence ofmagnesium are similar. Since metal ions have a profound effecton thestructureoftheHollidayjunction, it appearsthatthestructureofthebranchpointplaysakeyrole indeterminingtherateofspontaneousDNA branchmigration. We discuss the role of proteins in promoting the branch migrationstepduringhomologous recombination. Genetic recombination involving the exchange of genetic informationbetweentwoDNA moleculeshasbeenobserved invirtuallyallorganisms.An importantintermediateinboth homologousand site-specificrecombination istheHolliday junction,thebranchpointconnecting two duplexDNAs that are undergoingrecombination. Ifthebranchpoint isflanked by DNA sequence homology, the Hollidayjunction can spontaneouslymigrateineitherdirectionbytheexchangeof hydrogen bonds between the bases in homologous DNA strands.Thisprocess isknownas branchmigrationandis an important step in genetic recombination. In homologous recombination, the extent ofbranch migration affects the amount ofgenetic information thatistransferred between two homologues. Insite-specificrecombination, such as in theintegrationofbacteriophageA intotheEscherichia coli chromosome, branch migration inthe regionofhomology sharedbetweendonorandrecipientsitesisa prerequisitefor subsequentstepsintherecombinationpathway. Earlyestimatesoftherateofbranchmigrationyieldeda steprateofseveralthousandbasespersec (1),a ratethat was robustenoughtobeaccommodated withinthetimescaleof recombinationinbacteriophages.Thissuggestedthatbranch migrationcould occur spontaneouslyduringrecombination withouttheneedforproteinsto facilitatethisstep. Later, severalgroups studyingcruciformtransitionsinsupercoiled...
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PNAS-1994-Panyutin-2021-5_001 - Proc.Nat!.Acad. Sci. USA

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