ch12 dna replication and recomb

dna replication is semiconserv

meselson and stahl, use 2 isotropes of nitrogen. with equilibrium density gradient centrifugation

modes of replication

segment of dna undergoing replication = replicon
each replicon = ori of replication

theta replication

circular dna in e. coli
generates intermediate structure resembling theta!
double strand dna undwind at oriR, produce snigle nucleotide strands as templates for new dna synt
unwind of double helix makes loop/rep bubble
unwinding at one/both ends, making larger
dna rep is simultaneous with unwinding
point of unwinding where strands separate from double stranded dna helix = rep fork
2 forks, procede in bidirectional until meeting
produce 2 complete circular dna molecules, one old and one new

evidence: hot radioactive and cold nonradio strand

rolling circle replication

viruses, f factor of e.coli
initiate by break in one of nucleotide strands, exposing 3’-oh group and 5’ phosphate. new nucleotide adde to 3’ end of broken, using inner unbroken as template
5’ broken strand is displaced, rolling outlike a spool
3’ end grows around circle
rep fork may continue around number of times to produce several copies
each revolution - grow 3’ end displaces strand synt previous
eventually linear dna cleaved, double stranded circular dna and single strand linear dna, can serve as template for synt of complementary strand

linear eukaryotic replication

multiple oriR
dna unwinds at each with rep bubble
rep on both strands at each end of bubble
two rep forks spread outward
rep forks run in, replicon fuse
two identical dna molecule

summary of theta/roll circle and linear eukary rep

theta: circlar, no breaking of nucleotide strand, 1 replicon, both uni and bidirectional, two circular molecules
rolling circle: circular, yes, 1, uni, one circular and one linear that may circulize
linear eukary: linear, no, many, bi, two linear

requirements of replication

1. template of single strand dna: semiconserve so must unwind to expose bases that act as template for assembly of new polynucleotide strand, complementary and antiparallel to template

2. raw material/substrate to be assemble into new nucleotide strand: dNTP, 3-oh grup of last nucleotide on strand attack 5’ phosphate of dNTP cleaving 2 phosphate off and create phosphodiester bond btwn 2 nucleotide

3. enzyme and other protein that read the template and assemble substrate into dna molecule

direction of replication

new nucleotide joined to 3’ end of new synt strand
dna polymerase, enzyme that synt dna, add nucleotide only to 3’ of growing, so new always 5’ to 3’
two single strand dna temps are antiparallel and strand elongation always 5’ to 3’ , if going right to left than other left to right.
dna unwind during rep, antiparallel means one is expose in 5’ to 3’ and other is 3’ to 5’

continuous replication

as dna unwinds, template exposed 3’ to 5’ allows new strand to be synt continuously, in 5’ to 3’, leading strand

discontinous replication

other template is expose in 5’ to 3’, after short length of dna unwound, synt proceeds opposite of unwinding, because only short length need to be unwound to start, replication runs out of template. more dna unwound, so new template at 5’ end, so start anew and proceed again and again. short discontinuous bursts. lagging strand

bacterial replication stages

initiatioin, unwinding, elongation, termination

bacterial rep initiation

circular chromosome of e. coli has single oriC
initiator proteins (DnaA) bind to oriC and cause short section of DNA unwind
unwind allows helicase and single strand bind proteins SSB to attach to polynucleotide

bacterial rep unwinding

dna synt needs single strand template. so double strand dna has to be unwound. so cell needs several proteins and enzymes

dna helicase: break hydrogen bonds between bases, in bacteria cant initiate unwinding, so initiator protein separates dna strands at origin = short stretch of single strand dna, then load helicase onto single strand dna at each rep fork, once bound, move in 5’ to 3’ also moving rep fork

SSB: after unwound by helicase, SSB attach tightly to expose snigle strand dna. protect single strand and prevent formation of secondary structures like hairpins that coukld interfere w rep/ indifferent to base sequence, bind to any single strand dna. form tetramers of 4

dna gyrase: topoisomerase. control supercoil of dna. type 1 alter supercoil by making single strand break. type 2, gyrase, make double strand break. reduce torsional strain or torque buildign up ahead of rep fork as a result of unwinding. double strand break in one segment of dna helix, pass another segment of helix thru break, reseal broken ends of dna. requires atp, removes twist in dna and reduce supercoil. extra: antibiotics 4 quinolones kill bacteria by binding and inhibiting!

bacterial rep elongation

single strand dna used as template for synt of dna, requires series of enzymes

all dna polymerase need nucleotide with 3’-oh group to add new. cant initiate dna synt on bare template, need existing 3’-oh group

primers: synt short stretches of rna molecules, provide 3’-oh group so dna polymerase can attach dna nucleotide. but primer itself is rna polymerase so doesnt need itself. all new dna molecules have short rna primers and are remove later and replace with dna nucleotide

on leading strand at rep fork, where dna is continuous, primer required only at 5’ end of new synt strand. on lagging, primer at each okazaki fragment. forms complex with helicase at rep fork and moves along template of lagging. single primer on leading prolly synt by primase-helicase complex on template of lagging of other rep fork at opp end of rep bubble

dna synt by dna polymerase: e. coli has 5. 1 and 3 carryout dna synt in replication and other 3 are for dna repair.
3: replication. synt dna adding new nucleotides to 3’ end of growing dna strand. 5’ to 3’ polymerase allows it to add new nucleotides. 3’ to 5’ exonuclease removes nucleotides in that direction to correct errors. high processivity, capable of adding many nucleotides to growing dna strand without releasing. because of beta sliding clamp, encircles dna, allow polmerase slide easily. add dna nucleotide to primer to synt dna of lead and lag
1: 5’ to 3’ poly and 3’ to 5’ exo. synt dna and correct errors. also has 5’ to 3’ exo to remove primer and replace with dna. after 3, 1 remove rna and replaces with dna. lower processivity than 3.
but all 5 synt any sequence by template, synt in 5’ to 3’ by add nucleotides to 3’-oh, use dNTP to synt new dna, require 3’-oh to initiate synt, catalyze phosphodiester bond in dNTP, produce new synt strands complementary and antiparallel to template

ligase: break and nicks remain in sugar phsphate backbone of new dna strand because 3’oh group from dna poly 1 not attach to 5’ from dna poly 3. so ligase seals, catalyzes formation of phosphodiester bond without adding another nucleotide.

at rep fork: two units of dna poly 3 at rep fork one for each strand. connected. lagging loops so in psition for 5’ to 3’. dna poly 3 carry out 5’ to 3’ sim. on both, even tho opposite. requires 5
1. helicase to unwind dna
2. ssb protein to protect single nucleotide strands and prevent secondary structures
3. dna gyrase to remove strain ahead of rep fork
4. primase to synt primers with 3’oh group at beginning of each dna fragment
5. dna poly to synt lead and lag strand

bacteria e.coli dna poly summary

dna polymerase	5' to 3' poly	3' to 5' exo	5' to 3' exo	function
1	yes	yes	yes	remove and replace primer
2	yes	yes	no	dna repeair, restart rep after damage dna halts synt
3	yes	yes	no	elongate dna
4	yes	no	non	dna repair
5	yes	no	no	dna repair, translesion dna synt

bacterial rep components summary

initiator protein: binds to origin and separates strands of dna to initiate replication
dna helicase: unwinds dna at replication fork
ssbprotein: attach to single strand dna and prevent secondary structure from forming
dna gyrase: move ahead of rep fork, making and resealing breaks in double helical dna to release torque that builds as a result of unwinding at rep fork
dna primase: synt short rna primer to provide 3’ oh group for attachment of dna nucleotide
dna polymerase 3: elongate new nucleotide strand from 3’oh group provided by primer
dna poly 1: remove rna primer and replace with dna
dna ligase: join okazaki fragment by sealing breaks in sugar phosphate backbone of new synt dna

bacterial rep termination

rep terminated whenever 2 forks meet.
others specific term sequence or ter site block further rep
termination protein, tus in e.coli, binds to sequence. tus-ter complex that blocks movement of helicase. stalls rep fork. prevent further dna rep. bocks rep fork moving in one direction but not other

fidelity of dna rep

dna poly very particular. errors fixed by proofreading.

also mismatch repair: correct error after rep is complete. incorrect pair nucleotide remaining after rep produce deformity in secondary structure of dna. deformity recognize by enzyme, excise incorrect pair nucleotide. use og nucleotide strand as template to replace incorrect. requires ability to disgintuish between old and new dna. so methyl groups are added ater rep. old dna strand is methlated and distinguished. so mismatch repair on unmethylated

how is eukaryotic dna rep diff to bacterial

much greater size
multiple origins
chromosomes linear
dna te,plate assosicated with histone proteins in form of nucleosome, assembly must immediatel follow da replication

eukaryotic origin of rep

autonomoisly replicated sequences ARS enable any dna attach to to replicate
origins of rep in yeast
vary greatly in sequence
usually contain a-t base pairs
origin recognition complex ORC serve as inhibitor by bind to defined origins and initiate rep
many more potential rep origins than actually used during dna synt
role of initiator: orc recruits and loads helicase during g1 and during s helicase is activated and begins separating double strand dna into single strand templates used in dna synt

eukaryotic dna synt and cell cycle

replication is coordinated with cell cycle

controlled by cell cycle checkpoints

g1/s checkpoint: holds cell in g1 until dna ready to be replicated

cell enters s and dna is replicated

replication liscensing system ensures dna not replicated again until after cell pass thru mitosis

licensing of dna rep

1. origins are liscensed and approved for replocation. takes place early in cell cycle. replication licensing factors attach to each origin

2. replication machinery initiates rep at each lisense origin. function only at origin and occur early in cycle

occur in g1 of interphase when ORC binds to orgiin. orc + 2 addition lisensing factors allows MCM2-7 to bind. then in s, MCM2-7 assosicate with several cofactor, form active helicase to unwind double strand dna for rep. after rep begun, several mechs prevent mcm2-7 to bind to dna and reinitate rep at origin until after mitosis complete

eukaryotic rep unwinding

helicase that separate double strand dna isolated from eukary cells and ssb protein and topoisomerase

toposiomerase also need to remove supercoiling built up ahead of rep fork. clamp tight to dna and break one or both strands. strands revolve around each other. remove supercoil and strain. after dna relax, topoisomerase reseal broken ends of dna strands

cancer: anticancer drugs interferer with topoisomerase. blocking from researling broken ends of dna so cant remove supercoils ahead of rep so halt rep achinery and prevents proliferation of cancer cells. but also ihibits rep of normal noncancer cells. chemotherapy makes lot o people sick

eukaryotic dna polymerase

hellllllla more dna poly than bacteria. function in rep, recomb, dna repair

dna poly a, s, e.

a: primase activity, initiate dna synt by synt rna primer with short string of dna poly

after, s: complete rep on lagging strand

e: similar to s, rep leading strand. other poly help with repair and recomb or catalyze rep

s and e high speed rep and high fidelity, active sites snugly and exclusively accomadate 4 normal dna nucleotide ACTG

eukaryotic rep and nucleosome

histone protein to form nuclepsome. contribute to stability and packing of dna

quickly reasseble on to two new dna molecules.

1. disruption of og nucleosome on parent dna ahead of rep fork

2. redistribute preexisting histones on new dna molecule

3. addition of newly synt histones to complete form of new nucleosome

single da molecule associated with histone protein. after rep and nucleosome assembly, 2 dna molecules associate w histone protein. new octamers have mix of old and new histone. facilitate by histone chaperones, associate with helicase. accept old histone from og dna and deposit with new histone on 2 new dna.

eukary rep location in nucleus

polermase is fixed in ocation and template dna is threaded through

new synt dna emerge from other end

most rep in nucleus takes place at limited number of fixed sites = rep factory

new dupe dna extruded from these sites

eukary rep ends of chromosomes

linear and have ends

3’oh group needed by dna poly provided at initiation of rep by rna primers synt by primase.

solution is temporary!

eventually primers must be removed and replaced with dna nucleotide.

in circurlar, elongation provide 3'oh group immediately in front of primer so nucleotides can be added.

but for eukaryotes we have a problem!

3’oh griup preceding each primer. at very end, no adjacent stretch of rep dna to provide this. so when terminal primer remove, cnt be replaced by dna. removal produces gap at end of chromosome. so tchromsome would get shorter with each round of rep and self destruct

this problem assumes terminal primer located at very end. some yes but not for complex. and human ends shortern a lot faster if only terminal wasnt replaced. in human, 70-100 nucleotides from end.

telomeres: ends of chromosome. presence of many copy of short repeat sequence. TTAGGG in humans. usually the one with GGG excedes beyond the complement with C. g rich can be extended by telomerase, has protein and rna component. rna has 15-22 complementary nucleotide to g strand, so pairs with overhang and provide template for synt of additional dna. dna added to 3’ of g rich strand one at a time and rna template moves down. so telomerase can extend 3’ without complementary dna template. but we dk about the complementary C.

telomerase is in single cell eukary, germ cells, early embrynoic cells, and certain proliferative somatic cells (bone marrow and intestine) all need continuous cell division. but somatic have little or no, so they shortern every division. and only capanle of limited number of divisn. when telomers short beyond citical pt, chromosome unstable, rearragne, degrade, die

shortening of telomere = aging. somatic that express telomerase dont shorten and inhibi cell aging and divide indefinitely.

replication in archaea

common to eukary

proteins similar to eukary than bacteria

some single ori of rep, a lot have multiple

but fewer than eukary

dont have typical sequences recognizde by bacteria. instead similar to eukary

initiaor protein of archaea similar to eukary than bacteria

archaea closer to eukary than bacteria

recombination

exchange is between homologus dna is homologous recomb

takes place in crossing over

no genetic info lost or gained

pair of complementary bases. single nucleotide strand of chromosome pairs with complementary of another to form heteroduplex dna, consist of strands from diff sources

models of recomb

1. single strand break in each of two dna molecules, includes formation of holliday junction. double strand dna molecule of 2 homologous chromosome align. single strsand break in each dna molecule provide free end, invade, join free end of other dna molecule. strand invasion/joining on both dna molecule, 2 heteroduplex dna, consist of og strand and new strand from other dna molecule. pt where nucleotide strands pass is jolliday junction. it moves along molecules in branch migration. exchange prodce holliday intermeidate. can be cleaved horizontally and rejoin strands producing noncrossover recomb, genes on 2 ends indentical to og. cleavage in vertical and rejoin produce crossover recomb, genes on 2 ends are diff than og present

2. diuble strand breaks in one of two align dna molecule. removal of some nucleotide of end of broken strand, follow by strand invasion, displacement, replication. produce 2 heteroduplex dna molecule join by 2 holiday junction. interconnected molecule produce can be separated by further cleavage and reunion of nucleotide strands in same way holliday intermiediate separate in single strand break model. whether crossover or noncrossover molecule produce depend on whether cleavage is vert or horizontal

enzyme for recomb

unwind dna helices

cleave nucleotide strands

strand invasion

branch migration

further cleavage and union to remove holliday junction

e. coli recomb. recB C and D to form recBCD protein. unwind double strand dna and capable of cleave nucleotide strand

recA encode reca protein, allow invasion of dna helix by single strand and displacement of og strand

ukary: form and branch migration of holliday structure faciliated by enzyme Rad51

ruvA and B genes encode protein that catalyze branch migration. ruvC gene produce protein called resolvase, cleave holliday structure. cleav/resolution of structures in eukary carry out by analogous enzyme GEN1. SSB, dna ligase, dna polymerase, dna gyrase also