Chapter 10-First Part

Chap Ch apte terr 10: 10: DNA DNA REPL REPLIC ICA ATION ION AND RECOMBINATION  Three models of Replication 1. Semico Semicons nserv ervati ative ve repli replicat cation ion  Proposed in 1953 by Watson and Crick  Duri During ng DNA DNA repl replic icat atio ion, n, a doub double le stranded DNA molecule separate, and each strand is used as a template or  the synthesis o a ne! strand"  #his #his result results s in the ormat ormation ion o t!o t!o ident identica icall copies o the original double stranded molecule"

2. Cons Conser erva vati tive ve Repli Replicat catio ion n  Comp Comple leme ment ntar ary y poly polynu nucl cleo eoti tide de chai chains ns are are synthesi$ed"  %ollo! %ollo!ing ing synthe synthesis sis,, the t!o t!o ne!ly ne!ly create created d strands come together and the Parental strands reassociate  &riginal heli' is C&N()*+)D

*esearcher *esearchers s !ere !ere able to monitor monitor the process o replication by labelling DNA !ith '-th&midine, '-th&midine , a radioacti-e precursor o DNA and orming autoradiography" ("toradio$raph&   A techni;ue that pinpoints the location o a radioisotope in a cell" aylor and colleagues colleagues gre! root tips  #aylor or or
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3. Disp Disper ersi sive ve Rep Repli lica cati tion on  Parent Parental al strand strands s are disper dispersed sed into into t!o t!o ne! double helices ollo!ing replication"  )ach strand consists o both old and ne! DNA" n-ol-e clea-age clea-age o the parental strands during  n-ol-e replication"  .ost comple' and least likely to occur" Meselson-Stahl Experiment  .atthe! .eselson / %ranklin (tahl 01952"   An e'periment pro-iding a strong e-idence that semiconser-ati-e replication is the mode used by bac bacter terial ial cel cells to pro produc duce ne! DNA DNA molecules"  re! E. coli cells cells or many generati generation on in a medium !ith N4 ₄Cl as the only nitrogen source sed an isot isotop ope e o nitr nitrog ogen en to dist distin ingu guis ish h  sed bet!e bet!een en parent parent and ne!ly6 ne!ly6cop copied ied DNA" DNA" #he isotope o nitrogen had an e'tra neutron in the nucleus, !hich made it hea-ier7 N-1  Contains more than one neutron than the naturally occurring N618 isotope"  (table unlike radioacti-e isotopes   Ater many generations in the medium, almost all nitrogen6containing bases o DNA contained the hea-ier isotope" Sedimentation E!"ili#ri"m Centrif"$ation  Also called uoyant density gradient centriugation  (ample (amples s are orced orced by C)N#* C)N#*% %A A# #&N &N through a density gradient o a hea-y metal salt, Cesi"m Chloride. Semiconservative Semiconservative replication E"%ar&otes  :" 4erbert #aylor, Philip Woods, Walter 4ughes  )'perimented !ith root tips o the broad bean Vicia faba, !hich are e'cellent source o di-iding cells" 

)ro%ar&otic DN( Replication  DNA replication begi egins at the ori$ ori$in in of  replication and is is #idirectional rather rather than "nidirectional.   A replicon is replicon  is the length o DNA that is replicated ollo!ing one initiation e-ent at a single origin" DNA *eplication in Prokaryotes7 " N#A#&N 1" n!inding o the heli'

>" *educing increased coiling generated during un!inding 3" (ynthesis o a primer or initiation " )=&NA#&N 8" %ormation o ne! complementary strands by DNA polymerase 5" *emo-al o the *NA primers ?" :oining o the gap6illing DNA to the ad@acent strand " #)*.NA#&N  :ohn Carin #racked the replication o E.coli using  radioacti-e precursors o DNA autoradiography"  Demonstrated that in E.coli   there is only one region oriC, !here replication is initiated"  the entire chromosome constitues one replicon 0 8"?.b2 DNA *)P=CA#&N7 N#A#&N 1. *n+indin$ of the helix  Dna( 6 binds to the origin o replication 0oriC2 and is responsible or the initial steps in un!inding the heli'"  Dna,  DnaC 6 proteins that urther open and destabili$ed the protein"  'elicase 6 en$ymes that disrupt the hydrogen bonds that hold the t!o DNA strands together in a double heli'"  Replication for% 6 region o un$ip DNA 2. Red"cin$ increased coilin$ $enerated d"rin$ "n+indin$ DN( $&rase 6 rela'ed super coiling a member o DNA topoisomerase 6breaks and reorms DNAs phosphate backbone ahead o the replication ork Sin$le Strand #indin$ protein SS,)s/ 6 bind to the single strand DNA to pre-ent heli' rom reorming 3. S&nthesis of a primer for initiation  DN( )rimase 6 en$yme that generates *NA se;uence that ser-es as starting point or primer  or synthesis o o the ne! DNA chain" DNA *)P=CA#&N7 )=&NA#&N

0. ormation of ne+ complementar& strands  DNA polymerase  synthesi$es the DNA complementary o both strands o parent DNA  DNA polymerase  6 binds to template DNA as part o multiprotein comple'B synthesi$e only in 5 to 3 direction" DN( )ol&merase 

)n$ymes !hich synthesi$e nucleotide chains

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Prokaryotes7 DNA polymerase ,, , + / +

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DN( )4MER(SE 56 556   and 555  can elongate an e'isting *NA strand 0called a primer2 but cannot initiate DNA synthesis" All three possess 3 to 5 e'onuclease acti-ity

(ince DNA strands are antiparallel, the polymerase unctions asymmetrically" &n the leading strand, DNA is synthesi$ed continuously  &n the lagging strand, DNA is synthesi$ed in short ragments called %asa%i fra$ments  *eplication ork . Removal of the RN( primers  DNA polymerase  uses its 5 to 3 e'onuclease acti-ity to remo-e the *NA primer and ills the gap !ith ne! DNA 7. 8oinin$ the $ap fillin$ DN( to the ad9acent strand  DNA ligase @oins the ends o &kasaki ragments together" DNA *)P=CA#&N7 #)*.NA#&N DNA replication ends !hen7  *each the end o the chromosome 

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#!o replication orks meet