Lecture PowerPoint to accompany
Fourth Edition
Robert F. Weaver
Chapter 20
DNA Replication I:
Basic Mechanism and
Enyzmology
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

• Double helical model for DNA includes the concept that 2 strands are complementary
• Each strand can serve as template for making its own partner
– Semiconservative model for DNA replication is correct
– Half-discontinuous (short pieces later stitched together)
– Requires DNA primers
– Usually bidirectional
20-2

Three methods of DNA replication were considered:
1.
Semiconservative
2.
Conservative
3.
Dispersive
20-3

• DNA replicates in a semiconservative manner
• When parental strands separate
– Each strand serves as template
– Makes a new, complementary strand
20-4

• DNA replication in E. coli is semidiscontinuous
• One strand is replicated continuously in the direction of the movement of the replicating fork
• The other strand is replicated discontinuously as 1-2 kb Okazaki fragments in the opposite direction
• This allows both strands to be replicated in the 5’  3’-direction
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20-6

• Okazaki fragments in
E. coli are initiated with RNA primers 10-
12 nt long
• Intact primers are difficult to detect in wild-type cells because of enzymes that attack RNAs
20-7

• The replication structure resembles the
Greek letter,

• DNA replication begins with the creation of a “bubble” – a small region where parental strands have separated and progeny DNA has been synthesized
• As the bubble expands, replicating DNA begins to take on the
 shape
20-8

• In DNA replication, replicating forks represent sites of DNA replication
• Is the process:
– Unidirectional – one fork moving away from the other which remains fixed at the origin of replication
– Bidirectional – two replicating forks moving in opposite directions away from the origin
• Origin of replication is the fixed starting point for
DNA replication
• Replicon is the DNA under the control of one origin of replication
20-9

• Most eukaryotic and bacterial DNAs replicate bidirectionally
• ColE1 is an example of a DNA that replicates unidirectionally
20-10

• Circular DNAs can replicate by a rolling circle mechanism
– One strand of a dsDNA is nicked and the 3’-end is extended
– This uses the intact DNA strand as a template
– The 5’-end is displaced
• Phage

X174 replication cycles so that when one round is complete a full-length, singlestranded circle of DNA is released
• Phage l
, displaced strand serves as the template for discontinuous, lagging strand synthesis
20-11

l
• As the circle rolls right
– Leading strand elongates continuously
– Lagging strand elongates discontinuously
• Uses unrolled leading strand as a template
• RNA primers for Okazaki fragment
• Progeny dsDNA produced grows to many genomes before one genome worth is clipped off
20-12

• Over 30 different polypeptides cooperate in replicating the E. coli DNA
• Examine the activities of some of these proteins and their homologs in other organisms
– Start with DNA polymerases – the enzymes that make DNA
20-13

E. coli
• There are 3 DNA polymerases, the enzymes that make DNA, found in E. coli :
– pol I
– pol II
– pol III
• E. coli DNA polymerase I was the first polymerase identified
• It was discovered in 1958 by Arthur
Kornberg
20-14

• DNA polymerase I (pol I) is a versatile enzyme with 3 distinct activities
– DNA polymerase
– 3’  5’ exonuclease
– 5’  3’ exonuclease
– Mild proteolytic treatment results in 2 polypeptides
• Klenow fragment
• Smaller fragment
20-15

Contains both: Polymerase and 3’  5’ exonuclease activity which serves as proofreading
– If pol I added wrong nt, won’t base pair properly
– Pol I pauses, exonuclease removes mispaired nt
– Allows replication to continue
– Increases fidelity of replication
20-16

• Wide cleft for binding to DNA between two a-helices like a hand
– One helix is part of the “fingers”
– Other helix serves as the “thumb” domain
– Between the helices lies a b-sheet, palm
• 3 conserved Asp residues
• Essential for catalysis
• Likely coordinate Mg 2+
• Polymerase activity is far separated from the exonuclease activity
20-17


• This activity allows pol I to degrade a strand ahead of advancing polymerase
• Removes and replaces a strand in one pass
• Basic functions are:
– Primer removal
– Nick repair
20-18

• Pol II activity is not required for DNA replication
• Pol I appears mostly active in repair
• Only pol III is required for DNA replication
– Pol III is the enzyme that replicates bacterial
DNA
20-19

• Pol III core is composed of 3 subunits:
– DNA polymerase activity is in the

-subunit
– 3’  5’exonuclease activity found in 
-subunit
– Not yet clear what is the role of

-subunit
• DNA-dependent ATPase activity is located in the g
-complex containing 5 subunits
• Lastly, b
-subunit plus the other 8 comprise the holoenzyme
20-20

• Faithful replication is essential to life
• DNA replication machinery has a built-in proofreading system
– This system requires priming
– Only a base-paired nucleotide can serve as a primer for pol III holoenzyme
– If wrong nucleotide is incorporated accidentally replication stalls until 3’  5’ exonuclease of pol III holoenzyme removes it
• Primers are made of RNA which may help mark them for degradation
20-21

Mammalian cells contain 5 different DNA polymerases
– Polymerases d and
 appear to participate in replicating both DNA strands
– Priming DNA synthesis is

-subunit role
– Elongating both strands is done by d
-subunit
20-22

• DNA replication assumes that the 2 DNA strands at the fork somehow unwind
• Does not happen automatically as DNA polymerase does its job
– 2 parental strands hold tightly to each other
– This takes energy and enzyme action to separate them
– Helicase that unwinds dsDNA at the replicating fork is encoded by E. coli dnaB gene
20-23

• Prokaryotic ssDNA-binding proteins bind much more strongly to ssDNA than to dsDNA
– Aid helicase action by binding tightly and cooperatively to newly formed ssDNA
– Keep it from annealing with its partner
• By coating ssDNA, SSBs protect it from degradation
• SSBs are essential for prokaryotic DNA replication
20-24

• Strand separation of DNA is referred to as
“unzipping”
– DNA is not really like a zipper with straight, parallel sides, actually a helix
– When 2 strands of DNA separate, rotate around each other
• Helicase could handle this task alone if DNA were linear, short
• Closed circular DNA present special problems
– As DNA unwinds at one site
– More winding must occur at another site
20-25

• A “swivel” in the DNA duplex called DNA gyrase
• Allows the DNA strands on either side to rotate to relieve the strain
• Gyrase belongs to the enzyme class topoisomerase
• These add transient singleor double-stranded breaks into DNA
• Serves to permit change in shape or topology
20-26

• Enzymes called helicases use ATP energy to separate the two parental DNA strands at replicating fork
• As helicase unwinds 2 parental strands it introduces a compensating positive supercoiling force
• Stress of this force must be overcome or DNA will resist progression of replicating fork
• This stress releasing mechanism is the swivel
• DNA gyrase acts as swivel b pumping negative supercoils into replicating DNA
20-27

• DNA can be damaged in many different ways, if left unrepaired this damage can lead to mutation, changes in the base sequence of DNA
• DNA damage is not the same as mutation though it can lead to mutation
• DNA damage is a chemical alteration
– Mutation is a change in a base pair
– Common examples of DNA damage
• Base modifications caused by alkylating agents
• Pyrimidine dimers caused by UV radiation
20-28

• Alkylation is a process where electrophiles:
– Encounter negative centers
– Attack them
– Add carbon-containing groups ( alkyl groups)
20-29

Alkylating agents like ethylmethane sulfonate (EMS) add alkyl groups to bases
– Some alkylation don’t change base-pairing, innocuous
– Others cause DNA replication to stall
• Cytotoxic
• Lead to mutations if cell attempts to replicate without damage repair
– Third type change base-pairing properties of a base, so are mutagenic
20-30

• Ultraviolet rays
– Comparatively low energy
– Cause a moderate type of damage
– Result in formation of pyrimidine dimers
• Gamma and x-rays
– Much more energetic
– Ionize molecules around the DNA
– Form highly reactive free radicals that attack
DNA
• Alter bases
• Break strands
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20-32

• UV radiation damage to DNA can be directly repaired by a
DNA photolyase
• Uses energy from near-UV to blue light to break bonds holding 2 pyrimidines together
20-33

• O6 alkylations on guanine residues can be directly reversed by the suicide enzyme, O6methylguanine methyltransferase
• This enzyme accepts the alkyl group onto one of its amino acids
20-34

• Percentage of DNA damage products that can be handled by direct reversal is small
• Most damage involves neither pyrimidine dimers nor O6-alkylguanine
• Another repair mechanism is required, excision repair is the process that removes most damaged nucleotides
– Damaged DNA is removed
– Replaced with fresh DNA
– Base and nucleotide excision repair are both used
20-35

Base excision repair (BER) acts on subtle base damage
– Begins with DNA glycosylase
• Extrudes a base in a damaged base pair
• Clips out the damaged base
• Leaves an apurinic or apyrimidinic site that attracts
DNA repair enzymes
– DNA repair enzymes
• Remove the remaining deoxyribose phosphate
• Replace it with a normal nucleotide
20-36

E. coli
• DNA polymerase I fills in missing nucleotide in
BER
• Base is removed the AP site remains – apurinic or apyrimidinic
• AP endonuclease cuts or nicks DNA strand
• Phosphodiesterase removes the AP sugar phosphate
• Pol I performs repair synthesis
20-37

• DNA polymerase b fills in the missing nucleotide
– Makes mistakes
– No proofreading activity
• APE1 carries out proofreading
• Repair of 8-oxyguanine sites in DNA is special case BER – 2 ways can occur
– A can be removed after DNA replication by a specialized adenine DNA glycosylase
– oxoG will still be paired with C and oxoG removed by another DNA glycoslyase, oxoG repair enzyme
20-38

• Nucleotide excision repair typically handles bulky damage that distorts DNA double helix
• NER in E. coli begins when damaged DNA is clipped by an endonuclease on either side of the lesion, sites 12-13 nt apart
– Enzyme system catalyzing nucleotide excision repair is excinuclease
• Allows damaged DNA to be removed as part of resulting 12-13-base oligonucleotide
20-39

E. coli
• Excinuclease (UvrABC) cuts either side
• Remove oligonucleotide 12-13 nt
• DNA polymerase I fills in missing nucleotides using top strand as template
• DNA ligase seals the nick to complete the task
20-40

• Eukaryotic NER uses 2 paths
• GG-NER (global genome)
– Complex composed of XPC and hHR23B initiates repair binding lesion in the genome
– Causes limited amount of DNA melting
– XPA and RPA are recruited
– TFIIH joins, 2 subunits (XPB, XPD) use helicase to expand the melted region
– RPA binds 2 excinucleases (XPF, XPG) positions for cleavage
– Releases damaged fragment 24-32 nt long
20-41

• TC-NER is very similar to GG-NER
• Except:
– RNA polymerase plays role of XPC in damage sensing and initial DNA melting
• In either type, DNA polymerase
 or d fills in the gap left by removal of damaged fragment
• DNA ligase seals the DNA
20-42

20-43

• dsDNA breaks in eukaryotes are probably most dangerous form of DNA damage
• These are really broken chromosomes
– If not repaired lead to cell death
– In vertebrates can also lead to cancer
• Eukaryotes deal with dsDNA breaks in 2 ways:
– Homologous recombination
– Nonhomologous end-joining
• Role of chromatin remodeling in dsDNA break repair
20-44

• This process requires Ku and DNA-PK cs which bind at DNA ends and lets ends find regions of microhomology
• 2 DNA-PK complexes phosphorylate each other and activates
– Catalytic subunit to dissociate
– DNA helicase activity of Ku to unwind DNA ends
• Extra flaps of DNA removed, gaps filled, ends permanently ligated
20-45

• 2 protein kinases, Mec1 and Tel1 are recruited to
DSBs
• They phosphorylate Ser129 of histone H2A in nearby nucleosomes
• Phosphorylation recruits chromatin remodeler
IN080 to the DSB
– Use DNA helicase activity to push nucleosomes away from DSB ends
– Forms ssDNA overhangs essential for recombination
• SWR1 shares components with IN080
– Replaces phosphorylated H2A with variant Htz1
20-46

• Mismatch repair system recognizes parental strand by methylated A in GATC sequence
• Corrects mismatch in progeny strand
• Eukaryotes use part of repair system
• Rely on different, uncharacterized method to distinguish strands at a mismatch
20-47

• Direct reversal and excision repair are true repair processes
• Eliminate defective DNA entirely
• Cells can cope with damage by skirting around it
– Not true repair mechanism
– Better described as damage bypass mechanism
20-48

• The gapped DNA strand across from a damaged strand recombines with normal strand in the other daughter DNA duplex after replication
• Solves gap problem
• Leaves original damage unrepaired
20-49

• Induce the SOS response
• This causes DNA to replicate even though the damaged region cannot be read correctly
• Result is errors in the newly made DNA
20-50

• Humans have relatively error-free bypass system that inserts dAMPs across from pyrimidine dimer
• Replicate thymine dimers correctly
• Uses DNA polymerase
 plus another enzyme to replicate a few bases beyond the lesion
• If DNA polymerase
 gene is defective,
DNA polymerase
 and others take over
20-51

• Errors in correcting UV damage lead to a variant form of XP, XP-V
• DNA polymerase
 is active on templates with thymidine dimers and AP sites
• The polymerase is not really error-free
• With a gapped template, it is one of least accurate template-dependent polymerases known
20-52