Transcript Biochemistry Lecture 21

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
DNA Polymerase – cont’d
• Can only add nucleotides to preexisting strand
– So problematic at beginning of repl’n
– Problem solved by synth of ….
DNA Polymerase – cont’d
• Primers (25-5)
– Synth’d by
specialized enzymes
– Nucleic acid
segments
complementary to
template
– Often RNA
– Have free 3’ –OH
that can attack
dNTP
DNA Polymerase – cont’d
• 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 –
st
1
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
–  nucleosomelike structure
Initiation – cont’d
•  Unwinding of
helix (at 3 13‘tide seq area)
– ~13 nucleotides
participate in
unwinding
– Requires ATP
– Requires HU
(histone-like
protein)
Initiation – cont’d
• 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
Initiation – cont’d
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 deoxynucleotides 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
Lagging Strand – cont’d
• 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
Lagging Strand – cont’d
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
Lagging Strand – cont’d
• 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
Lagging Strand – cont’d
– 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 a
• In nucleus
• Has subunit w/ primase activity
• May be impt to lagging strand synth
– DNA polymerase d
• Assoc'd w/ a
• Impt to attaching enz to nucleic acid chain
• Has 3'  5' exonuclease ability (proofreading)
– DNA polymerase e
• 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 Nglycosyl 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 sugarphosphate backbone
Fig.25-22