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Chapt 20 DNA Replication I:
Basic Mechanism and Enyzmology
Student learning outcomes:
• Describe general features of semi-conservative DNA
replication: leading, lagging, strands; requirement for
primers; bidirectional, rolling circle
• Describe DNA polymerases: general enzymology
and comparison of prokaryotes, eukaryotes
• Describe major types of DNA damage and repair:
Important Figures: 1, 4, 7*, 9, 10, 13, 14, 15, 16, 18, 22, 24,
26, 27, 28, 29, 30, 31*, 32, 33*, 34, 36, 37, 38, 39, 40, 41
Review problems: 8-11, 13-15, 22-24, 27-29, 32-35; AQ4
20-1
20.1 General Features of DNA Replication
• Double helical model for DNA: complementary strands
• Each strand is template for new partner strand
– Semiconservative model for DNA replication 5’ -> 3’
– Leading strand continuous synthesis
– Half-discontinuous (short pieces on lagging strand are
later stitched together)
– Requires RNA primers
– Usually bidirectional (bacterial and eukaryotes)
• Origin of replication (ori): fixed starting point
• Replicon: DNA under control of one ori
20-2
Semi-discontinuous Replication
• DNA polymerase only
synthesizes 5’3’ direction
• RNA primers 10-12 nt long
in E. coli
• Leading strand replicates
continuously in direction of
movement of fork
• Lagging strand replicates
discontinuously in direction
opposite to fork (as 1-2 kb
Okazaki fragments)
Fig. 7
20-3
Bidirectional Replication
• Replication structure resembles Greek letter, 
• DNA replication begins with creation of “bubble” –
small region where parental strands separated,
progeny DNA synthesized
• As bubble expands, replicating DNA is  shape
Fig. 9
20-4
Rolling Circle Replication
• Circular DNAs can replicate as rolling circle
– One strand of dsDNA is nicked, 3’-end extended (leading)
– Uses intact DNA strand as template
– 5’-end gets displaced; lagging synthesis fills in
• Phage l: leading strand elongates continuously;
• displaced strand serves as template for discontinuous, lagging
strand synthesis; can get many genome sized piece
Fig. 20
20-5
20.2 Enzymology of DNA Replication
• >30 different polypeptides to replicate E. coli DNA
• Biochemical purifications, conditional mutants to
examine activities of proteins (essential activities)
• DNA polymerases – enzymes that make DNA
– Require primer, Mg++, buffer, dNTPs
• 3 DNA polymerases in E. coli:
– pol I - repair enzyme
– pol II - non-essential
– pol III – the real replication enzyme
20-6
DNA Polymerase I
Fig. 16
• E. coli DNA polymerase I –
– first enzyme (1958, Arthur Kornberg)
• Pol I has 3 distinct activities:
– DNA polymerase
– 3’5’ exonuclease – proofreading
– 5’3’ exonuclease – degrades strand ahead of it
• Can remove primers
– Mild proteolytic treatment ->2 polypeptides
• Klenow fragment (lacks 5’->3’ exonuclease)
– Can fill in sticky ends left by restriction enzymes (Fig. 15)
– Steitz structure 1987
20-7
Pol III Holoenzyme
Pol III core has 3 subunits: catalysis, proofread,
Pol III g complex has 5 subunits – DNA-dependent ATPase
Pol III holoenzyme includes b subunit
Charles McHenry (CU SOM) biochemical studies
20-8
Eukaryotes have multiple DNA Polymerases
Mammalian cells 5 different DNA polymerases
– Polymerases d and a replicate both DNA strands
– PCNA factor helps with processivity
20-9
Other enzymes for replication E. coli
• Helicase – uses ATP to unwind strands
– Creates positive supercoils
– dnaB gene
• Single strand DNA binding protein – SSB
– Stimulates polymerization
• DNA gyrase (topoisomerase II)
– Negative supercoils, swivel
20-10
20.3 DNA Damage and Repair
• DNA can be damaged in many different ways:
– Cells have many ways to repair damage, easier to repair
before DNA is replicated
– if unrepaired, damage can lead to mutation
• DNA damage is not the same as mutation, but it
can lead to mutation
• DNA damage is chemical alteration
• Mutation is inherited change in base pair
– Common examples of DNA damage
• Base modifications caused by alkylating agents
• Pyrimidine dimers caused by UV radiation
20-11
Alkylation of Bases Causes Damage
• Alkylation - process
where electrophiles:
– Attack negative
centers
– Add carbon-containing
groups (alkyl groups)
Common targets (red):
N7 of G, N3 of A,
phosphodiester bond
O6 of G
Fig. 27
20-12
Alkylation of Bases Causes Damage
Alkylating agents like ethylmethane sulfonate (EMS):
– Some alkylations don’t change base-pairing – innocuous
– Others cause DNA replication to stall:
• Cytotoxic
• Mutations if cell attempts to replicate without repair
– Others change base-pairing properties, so are mutagenic:
• Ethyl O6-G mispairs with T -> GC ->AT transition mutation
Fig. 28
20-13
UV Radiation Damages DNA
• Ultraviolet rays (260 nm)
– Comparatively low energy
– Moderate type of damage
– Result in formation of
pyrimidine dimers
– Mostly T-T dimers
– T-T dimers distort DNA,
block replication and
transcription
Fig. 29: Thymine dimers
have cyclobutane ring
20-14
Ionizing Radiation Damages DNA
• Gamma and x-rays
C8->
– Much more energetic
– Ionize molecules around DNA
– Highly reactive free radicals
attack DNA
• Alter bases
• Break DNA strands
– Especially double strand
– (useful cancer therapy)
Fig. 30: oxidative damage
forms 8-oxo-guanine;
At replication, A often is
inserted opposite -> mutation
20-15
UV DNA Damage can
be directly reversed
• Photoreactivation (light repair)
• DNA photolyase uses energy
from near-UV to blue light to
break bonds holding 2
pyrimidines together
• Enzyme in most organisms (not
placental mammals)
Fig. 31
20-16
Reversing High Energy DNA Damage
• O6 alkylations on G residues directly reversed by
enzyme O6-methylguanine methyltransferase
• Enzyme accepts alkyl group onto SH of Cys - and is
inactivated (suicide enzyme)
• In E. coli, enzyme is induced by DNA alkylation
Fig. 32
20-17
Excision Repair
• Only small percentage of DNA damage products
are directly reversed
• Excision repair removes most damaged
nucleotides:
– Damaged DNA is removed
– Replaced with fresh DNA
– Both base and nucleotide excision repair
20-18
Base Excision Repair
(BER): specific enzymes
remove damaged base
DNA glycosylase
• Extrudes base in damaged
base pair, and clips out
• Leaves apurinic or
apyrimidinic (AP) site that
attracts DNA repair enzymes
DNA repair enzymes
• Remove remaining
deoxyribose phosphate
• Replace with normal
nucleotide, ligate
Fig. 33
20-19
Eukaryotic BER
Fig. 34
• DNA polymerase b fills in
missing nucleotide
Makes mistakes, not proofread
• APE1 proofreads
– (AP endonuclease)
• Repair of 8-oxoG sites is special case of BER:
– After replication, A often is inserted; A can be removed by specialized
adenine DNA glycosylase
– Before replication, oxoG paired with C; the oxoG is removed by oxoG
20-20
DNA glycoslyase (hOGG1)
Nucleotide Excision Repair (NER)
• NER handles bulky damage that distorts DNA
– Including Thymine dimers, large adducts
• Specific endonucleases clip DNA strand on either
side of lesion, remove single strand, resynthesize
and rejoin.
• Xeroderma pigmentosum
(XP) people have hereditary
increased skin cancer;
lack NER enzymes
20-21
NER in
E. coli
Fig. 36
•
•
•
•
Excinuclease (UvrABC) cuts either side of damage
Remove 12-13 nt oligonucleotide
Pol I fills in using top strand as template
DNA ligase seals nick
20-22
Eukaryotic NER uses 2 paths
• GG-NER
–
–
–
–
–
–
Complex of XPC and hHR23B initiates repair, binds lesion
limited DNA melting
XPA and RPA recruited
TFIIH joins, helicase expands melted region
RPA binds 2 excinucleases (XPF, XPG) cleaves
Releases damage 24-32 nt
• Transcription-Coupled (TC-NER) resembles GG-NER
Except:
– RNA polymerase plays role of XPC in damage sensing and
initial DNA melting
20-23
Human Global Genome NER
• Complex of XPC and
hHR23B initiates
repair, binds lesion
• Limited DNA melting
• XPA + RPA recruited
• TFIIH joins, helicase
expands melted region
• RPA binds 2
excinucleases (XPF,
XPG),
• Cleaves, releases
damage 24-32 nt
Fig. 37
20-24
Transcription-Coupled (TC)-NER
Resembles GG-NER:
– RNA
polymerase
plays role of
XPC in damage
sensing and
initial DNA
melting
– RNAP stalls
Fig. 37
20-25
Double-Strand Break (DSB) Repair in
Eukaryotes
• dsDNA breaks in eukaryotes are very dangerous
• Broken chromosomes
– If not repaired, lead to cell death
– In vertebrates, also leads to cancer
• Eukaryotes deal with dsDNA breaks in 2 ways:
– Homologous recombination with good chromsome
– Nonhomologous end-joining (NHEJ) has errors
– Chromatin remodeling has role in dsDNA break repair]
[
20-26
Model for
Nonhomologous EndJoining
• Ku and DNA-PKcs bind at DNA
ends and let ends find
microhomology
• 2 DNA-PK complexes
phosphorylate each other:
– Catalytic subunit dissociates
– DNA helicase activity of Ku unwinds
DNA ends
• Extra flaps of DNA removed,
gaps filled, ends ligated
• Inaccurate process, DNA is lost
Fig. 38
20-27
Mismatch Repair
• Recognizes parental DNA
by its methylated A in
GATC sequence (E. coli)
• Corrects mismatch in
progeny strand
• Eukaryotes use part of
repair system – unclear
how distinguish strands at
mismatch
• HNPCC colon cancer- defects in
repair of mismatch damage
cause instability of microsatellite
regions, many mutations
Fig. 39
20-28
Coping with DNA Damage Without
Repairing It
• Direct reversal and excision repair are true repair
processes - accurate
• Eliminate defective DNA entirely
• Cells also copes with damage by skirting it
– Not true repair mechanism
– Damage bypass mechanism:
• gives time to repair
• cell can replicate, fix damage later
20-29
Recombination Repair
• Gapped DNA strand across
from damaged strand
recombines with normal
strand in other daughter DNA
duplex after replication
– Must occur before segregation
• Solves gap problem
• Leaves original damage
unrepaired – fix later
Fig. 40
20-30
Error-Prone
Bypass (SOS)
•
•
•
•
Induce SOS response
Activates recA protease
UmuC/D dimer is DNA pol V
Causes DNA to replicate
even though damaged
region not read correctly
• Errors in newly made DNA,
but cell lives
Fig. 41
• Mutants of umu genes die,
but do not have mutations
Recall, UV damage to cell can
induce SOS path, which causes
cleavage of lambda repressor,
return of prophage to lytic cycle
20-31
Error-Prone Bypass in Humans
• Humans have relatively error-free bypass system
that inserts dAMPs across from pyrimidine dimer
• Specialized DNA polymerases are activated
• Replicate thymine dimers correctly
• Uses DNA polymerase  plus another enzyme to
replicate a few bases beyond lesion
– Polymerase is not really error-free
• If DNA polymerase  gene is defective, DNA
polymerase  and others take over, more errors
20-32
Review questions
• 8. Diagram rolling circle replication of lambda
• 12, 16. List the different DNA polymerases in E.
coli and eukaryotes and explain their roles.
• 24. Compare/ contrast base excision repair and
nucleotide excision.
• 33. Diagram recombination repair in E.coli.
20-33