DNA Replication

DNA Replication

Chapter 25

DNA Polymerase (E. coli ex)

• Catalyzes synth of new DNA strand

• d(NMP)n + dNTP d(NMP)n+1 + PPi

• 3’ –OH of newly synth’d strand attacks first phosphate of incoming dNTP

• Rxn thermodynamically favorable

–Why??

DNA Polymerase – cont’d

• Noncovalent stabilizing forces impt

– REMEMBER??

• Base stacking hydrophobic interactions

• Base pairing multiple H-bonds between duplex strands

– As length of helix incr’d, # of these forces incr’d incr’d stabilization


• Can only add nucleotides to pre-existing strand

– So problematic at beginning of repl’n

– Problem solved by synth of ….


• Primers (25-5)

– Synth’d by specialized enzymes

– Nucleic acid segments complementary to template

– Often RNA

– Have free 3’ –OH that can attack dNTP


• Once DNA polymerase begins synth of DNA chain, can dissociate OR can continue along template adding more nucleotides to growing chain

– Rate of synth DNA depends on ability of enz to continue w/out falling off

– Processivity

DNA Polymerase – Accuracy

• Enz must ACCURATELY add correct nucleotide to growing chain

– E. coli accuracy ~ 1 mistake per 109 – 1010 nucleotides added

• Geometry of enz active site matches geom. of correct base pairs (25-6)

– A=T, G=C fit

– Other pairings don’t fit

Fig.25-6

Accuracy – cont’d• Enz has “back-up” proofreading ability

– Its conform’n allows recognition of improper pairing

– Has ability to cleave improperly paired bases (25-7)

• Called 3’ 5’ exonuclease activity

• Enz won’t proceed to next base if previous base improper

– Then catalyzes add’n of proper base

– Increases accuracy of polymerization 102 – 103 X

• Note: cell has other enz’s/mech’s to find/repair mistakes (mutations) after new helix synth’d w/ repl’n

Fig.25-7

3 E. coli DNA Polymerases (Table 25-1)

• I -- impt to polymerase activity

– Slow (adds 16-20 nucleotides/sec)

– Has 2 proofreading functions

– Only 1 subunit

• II – impt to DNA repair

– Less polymerization activity

– Several subunits

3 DNA Polymerases -- cont’d

• III – principle repl’n enz

–Much faster than polymerase I (adds 250-1000 nucleotides/sec)

–Many subunits (Table 25-2) each w/ partic function

• Encircles DNA; slides along helix (25-10)

• One subunit “clamps” helix better processivity

Fig.25-10

Replisome• Many other enz’s/prot’s necessary for repl’n

(Table 25-3)

• Complex together replisome

• Helicases – sep strands

• Topoisomerases – relieve strain w/ sep’n

• Binding proteins – keep parent strands from reannealing

• Primases – synth primers

• Ligases – seal backbone

– What bonds hold nucleic acid backbone together?

Initiation – 1st Stage Repl’n• E. coli unique site = ori C (25-11)

– 3 adjoining 13-nucleotide consensus seq’s

– Non-consensus “spacer” nucleotides

– 4 9-nucleotide consensus seq’s spaced apart

• Consensus seq’s contain nucleotides in partic seq common to many species

Initiation – cont’d

• At ori C (at 4 9-‘tide seq area) (25-12)

– ~20 DnaA mol’s (proteins) bind

– Requires ATP

nucleosome-like structure


Unwinding of helix (at 3 13-‘tide seq area)

– ~13 nucleotides participate in unwinding

– Requires ATP

– Requires HU (histone-like protein)


• Unwound helix is stabilized

– Requires DnaB, DnaC (proteins)

• These bind to open helix

– DnaB also acts as helicase

• Unwinds DNA helix by 1000’s of bp’s


Result:

– Nucleotide bases now exposed for base pairing in semiconservative repl'n

• What does semiconservative mean?

– Yields 2 repl’n forks

Initiation – cont’d• Other impt repl’n factors at repl’n forks

(Table 25-4)

– SSB = Single Strand DNA Binding Protein

• Stabilizes sep’d DNA strands

• Prevents renaturation

– DNA gyrase -- a topoisomerase

• Relieves physical stress of unwinding

• Note: in E. coli, repl’n is regulated ONLY @ initiation

Elongation

• Second stage of repl’n

• Must synth both leading and lagging strands

– REMEMBER: 1 parent strand 3' 5; its daughter can be synth'd 5' 3' easily. What about the other parent strand (runs 5' 3')??

• Follows init’n w/ successful unwinding repl’n fork, stabilized by prot’s

– So have parent strands available as templates for base-pairing 2 daughter dbl helices

Elongation -- cont'd – Leading Strand

• Simpler, more direct (25-13)

• Primase (=DnaG) synthesizes primer

– 10-60 nucleotides

– NOT deoxynucleotides

• Short RNA segment

– Occurs @ fork opening

– Yields free 3’ –OH that will attack further dNTP’s

Leading Strand – cont’d

• DNA polymerase III now associates

– Catalyzes add’n of deoxy-nucleotides to 3’ –OH (25-5)

Leading Strand – cont’d

• Elongation of leading strand keeps up w/ unwinding of DNA @ repl’n fork

– Gyrase/helicase unwind more DNA further repl’n fork

– SSB stabilizes single strand DNA til polymerase arrives

– Synth continues 5’ 3’ along daughter strand

Fig.25-13

Elongation -- cont'd – Lagging Strand

• More complicated

• REMEMBER: still need 5’ 3’ synth, AND still need to have antiparallel strands.

– Template strand here is 5’ 3’

– Can’t synth continuous daughter strand 5’ 3’

– Cell synth’s discontinuous DNA fragments (Okazaki fragments) that will be joined (25-13)

• Must have several primers AND coordinated fork movement

Fig.25-13

Lagging Strand – cont’d

• Lagging strand is looped next to leading strand (25-14)

– DNA polymerase III complex of subunits catalyzes nucleic acid elongation on both strands simultaneously

– Primosome = DnaB, DnaG (primase) held together w/ DNA polymerase III by other prot’s

Fig.25-14


• One subunit complex of DNA polymerase III moves along lagging strand @ fork in 3’ 5’ direction (along parent)

– Another subunit complex of polymerase III synth’s daughter strand along leading strand

• At intervals, primase attaches to DnaB (helicase)

– Here, primase catalyzes synth of primer (as on leading strand)

– Also (once primer synth’d), primase directs “clamp” subunit of polymerase III to this site

– This directs other polymerase III subunits to primer


Now polymerase III catalytic subunits add deoxynucleotides to primer Okazaki fragment

– Book notes primosome moves 3’ 5’ along daughter strand, but both primase & polymerase synthesize strands 5’ 3’ along daughter

Fig.25-14


• Okazaki fragments must be joined

– DNA polymerase I exonuclease cleaves RNA primer (25-15)

– DNA polymerase I simultaneously synth’s deoxynucleotide fragment

• 10-60 nucleotides

• Nicks between fragments


– DNA ligase seals nicks between fragments (25-16)

• Catalyzes synth of phosphodiester bond

• NADH impt (coordination role?)

Fig.25-16

Termination

• Repl'n has occurred bidirectionally @ 2 forks concurrently

• E. coli genome is closed circular

– So 2 repl'n forks will meet

Termination – cont’d

• Ter = seq of ~ 20 nucleotides (25-17)

• Tus = prot's that bind Ter

• When replisome encounters Ter-Tus

– Replisome halted

– Repl'n halted

– Replisome complex dissociates

Termination – cont’d

• Result = 2 intertwined (catenated) circles

– Topoisomerase IV nicks chains

– One chain winds through other

– 2 Complete genomes sep'd

Eukaryotic DNA Replication• Repl'n mechanism & replisome structures

similar to prokaryotes, BUT:

– DNA more complex

• Not all is coding for peptides

– Chromatin packaging more complex

• REMEMBER: nucleosomes, 30 nm fibers, nuclear scaffold, etc.

– No single origination pt for repl'n

• Many forks develop

Simultaneous repl'ns bidirectionally

• Forks move more slowly than in E. coli

– But efficient because more forks

Eukaryotic DNA Replication• Repl'n enzymes not yet fully understood

– DNA polymerase

• In nucleus

• Has subunit w/ primase activity

• May be impt to lagging strand synth

– DNA polymerase • Assoc'd w/

• Impt to attaching enz to nucleic acid chain

• Has 3' 5' exonuclease ability (proofreading)

– DNA polymerase • Impt in repair

Eukaryotic DNA Replication

• Replisome proteins not yet fully understood

– Found prot's similar to SSB prot's of E. coli

• Termination seems to involve telomerases

– Telomeres = seq's @ ends of chromosomes

DNA Alterations• Need unaltered, correct nucleotide seq to code

for correct aa's correct peptides correct proteins

– Some changes acceptable

• Some "wobble" in genetic code

– Some DNA damage in mature cells can be fixed

• DNA repair mechanisms avail for TT dimers (ex)

• Have (more) other mature cells that can maintain homeostasis in organism

• BUT -- if mispaired bases during repl'n mutation in daughter cell (and her subsequent daughters)

Definitions

• Lesion = unrepaired DNA damage

–Mammalian cell prod's > 104 lesions/day

• Mutation = permanent change in nucleotide seq

– Can be replicated during cell division

– Results if DNA polymerase proofreading fails

–May occur in unimpt region = Silent Mutation

• Doesn't effect health of organism

Definitions – cont’d

• Mutation -- cont’d

–May confer advantage to organism = Favorable

• Rare

• Impt in evolution

–May be catastrophic to organism health

• Correlations between mutations & carcinogenesis

DNA Repair

• Cell has biochem mech's to repair damage to DNA

– Though 104 lesions/day, mutations < 1/1,000 bp's

• If repair mech's defective disease/dysfunction

– Ex: xeroderma pigmentosum

• UV light DNA lesions

• No repair mech

Skin cancers

• Repair mech ex: base excision repair

– Takes advantage of complementarity of strands

Base Excision Repair

• N-Glycosylases

– Cleave N-glycosyl bonds

• What parts of nucleic acid are joined by N-glycosyl bonds?

– Several specific N-glycosylases

• Each recognizes a common DNA lesion

– Common -- bases altered by deamination events

– Yields apurinic or apyrimidinic (AP) site

Excision Repair – cont’d

• Uracil Glycosylase -- ex

– Deamination of cytosine uracil (improper)

– Enz recognizes, cleaves ONLY U in DNA

• Not U in RNA

• Not T in DNA

AP site on DNA (25-22)

• Would this be apurinic or apyrimidinic?

• Leaves behind sugar-phosphate of original nucleotide

Excision Repair – cont’d

– Then other enz's (AP endonucleases) cleave several bases of mutated strand around AP site

– Then DNA polymerase I catalyzes polymerization of proper nucleotides at site

– Then DNA ligase seals nicks on sugar-phosphate backbone

Fig.25-22

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DNA Replication