BIOL 3301 - Genetics Ch11A - Replication of DNA in Prokaryotes 08 St

8/18/2019 BIOL 3301 - Genetics Ch11A - Replication of DNA in Prokaryotes 08 St

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Semiconservative Replication

• Conservative, semiconservative and

dispersive recombination

• Semiconservative mechanism suggested by

Watson and Crick

• Confirmed by Matthew Meselson and

Franklin Stahl eperiments, !"#$


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%&' (olymerase + / E. coli0

• Single polypeptide !12,111 %a

• (roduct of gene pol A

• 3n*ymatic activity ) not true %&' replicase4 ) (olymerase activity #5to 25

) 3onuclease activity #5to 256 cuts back %&' strands

starting at #5 up to !1 nucleotides

) 3onuclease activity 25to #56 cleaves off

mononucleotides from 25


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7ther %&' (olymerases / E. coli0%&'

polymeraseFunction Structure 3n*ymatic

activity

++ %&' repair Single polypeptide

(olymerase #5to 25

3onuclease 25to #5

+++ replicase Comple, core )2 polypeptides

(olymerase #5to 25

3onuclease 25to #5

+ %&' repair

%&' repair


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%&' (olymerase +++ / E.coli0

• .rue replicase

• (roduct of polC gene / now dnaE 0

• Multimeric en*yme, "11,111 %a )holoen*yme /si polypetides0

• Minimal core with catalytic activity ) 2

subunits 8,9, :• 'dd ; subunit ) dimeri*ation of the

catalytic core, increased activity


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%&' (olymerase +++ / E.coli0

• eta subunit ) dimeric clamp, keeps itattached to %&' but allow to move easily,slide

• Synthesis of both strands at a replicationfork in E.coli re=uire ? @1 polypetides, products of at least A genes


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What 're .he .hree Stages of

%&'

ReplicationB• +nitiation ) unwinding of %&' heli and

stabili*ation ) starts at origin of replication,

re=uire comple of proteins, primosome• 3longation ) synthesis of new %&' strands )

semiconservative, bidirectional, leading

strand, lagging strand• .ermination ) formation of a newly

synthesi*ed strand, nucleoid formation


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+nitiation 7f Replication

• >acteria ) single point of replication, oriC

• oriC ) @# bp, repeating se=uences of "th

and !2th base pairs )"mers and !2mersD

• .he orientation, spacing, and se=uences of

the "6bp repeats are critical for function of

oriC


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7rigin of Replication

• 7rigins contain %&' se=uences recogni*ed

by replication initiator proteins 6 %na' in E

coli

• .hese initiator proteins recruit other

proteins to separate the two strands and

initiate replication forksD


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7rigins of Replication

• +nitiator proteins recruit other proteins to separatethe %&' strands at the origin, forming a bubbleD

• 7rigins tend to be E'.6rich to assist this process• 7nce strands are separated, R&' primers are

created on the template strands

• %&' polymerase etends these primers to create

newly synthesi*ed %&'


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7rigins of Replication

• 's %&' synthesis continues, the original %&'

strands continue to unwind on each side of the

bubble, forming replication forksD• +n bacteria, which have a single origin of

replication on their circular chromosome, this

process eventually creates a Gtheta structureG


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Replication in E. coli

• >egins at oriC

• Replication buble ) formed by interaction

of prepriming proteins with oriC

) %na' protein /product of dnaA gene0 binds to

four "mer repeats of oriC

) ' core of @161 polypeptides with oriC, %&'wound on proteins


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Hnwinding of %&'

• +nitiator protein binds to replicator /%na' protein, product of dnaA gene0 6" bp region

• Stimulates denaturing at '.6rich regions ) !2 bpregions

• %&' helicases /dnaB gene0 are loaded onto %&'

• >egin untwisting the %&' in both directions from

oriC ) two replication forks


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Initiation of DNA Replication

at oriC


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Hnwinding of %&'

• 3ach gyre /or turn0 is !1 nucleotides

• %&' molecule is rotated 2I1o once for each

!1 nucleotides replicated

) E.coli replication ) 21,111 nuclJmin

) Spin ) 2,111 revolutions per minute

• %&' helicases, product of dnaB gene,

re=uire '.(


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Hnwinding of %&'

• Single strand formed is covered with single

strand %&'6binding proteins ) SS>(, ssb

gene• Cooperative binding ) first bond stimulates

net ones


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Hnwinding of %&'

• 3ach %&' helicase recruits %&' primase

/dnaG gene0

• (rimosome is formed /helicase L primase0

• %&' primase synthesi*es a short R&'

primer ) #6!1 nt

• 7nly then %&' polymerase can start


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Replication in E. coli -

elongation• Semiconservative

• Re=uire %&' polymerase

• Continuous on one strand

• %iscontinuous on another strand

•7ccur in #5 to 25 direction only

• Re=uire free )7< group on 25 end

• Would not proceed without a primer


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Step @4 3longation

• For polymeri*ation activity, %&' polymerases re=uire4

) .riphosphate nucleosides d&.(s ) Mg @L

) .emplate, single stranded

) (rimer ) free 2567


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• SS> proteins keep strands seperated

• (rimosome produces R&' primers on both %&'

strands• %&' polymerase +++ starts elongation using R&'

primer

• %&' is made in opposite directions on both

strands ) Qeading strand

) Qagging strand


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Qeading and lagging strands

• Qimitation is imposed by synthesis of %&' at 25

end /#5 to 25 synthesis0

• %&' strands are used differently at replicationfork

) leading strand is used for continuous %&' synthesis

) lagging strand is used in discontinuous synthesis

• forms 7ka*aki fragments

• fragments Koined by %&' ligase leading

lagging


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•


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• %&' pol +++ leaves the region

• %&' pol + continue #5625 synthesis and

remove primer as #525 eonuclease

activity

• %&' ligase Koins the nicks


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Replisome

• Replisome ) replication apparatus moving along

%&' molecule at replication fork

• %&' polymerase +++ holoen*yme ) 7ne catalytic core replicates leading strand

) 'nother replicates lagging strand

) (rimosome unwinds %&' in leading strand and

synthesi*e primers

) Qagging strand forms a loop


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(roof6reading of Replication

• .hanks to the precision of the process

/which includes a Gproof6readingG function0,

the Kob is done with only about one incorrectnucleotide for every !1" nucleotides

insertedD

• +n other words, more often than not, the3Dcoli genome /DA!1I0 is copied without

error

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BIOL 3301 - Genetics Ch11A - Replication of DNA in Prokaryotes 08 St