Lecture 17

DNA replication
Table 11.1 Comparison of E. coli DNA polymerases

• Primary replicating enzyme in E. coli cells is thought

to be DNA pol III: faster, more complex structure.
• DNA pol I and II probably serve in editing and repair of
DNA.
A DNA Replication Paradox:

1. DNA strands must be copied in both directions during

replication.
2. However, all known DNA polymerases catalyze chain
formation in the 5’3’ direction.

Paradox resolved by Reiji Okazaki and coworkers:

• Observed short 1000-2000 nucleotide fragments
during replication.
• Small fragments are covalently joined in later steps to
form completed lagging daughter strand.
Semidiscontinous replication
• If DNA strands are extended 5’ to 3’, how can the
antiparallel strand by simultaneously replicated past
the replication fork?
• Okazaki used pulse-chase experiment.
• E. coli DNA pulse-labeled 30 s with of [3H]thymidine
that changes the sedimentation coefficient of newly
made DNA from 7S to 11S (Okazaki fragments).
• Okazaki fragments: 1000-2000 nt in prokaryotes; 100-
200 nt in eukaryotes
• Follow [3H]thymidine pulse with transfer to unlabeled
medium (pulse-chase)-the resulting radioactively
labeled DNA sediments at rate that increases with
time the cells grow in unlabeled medium.
• Okazaki fragments incorporated into the larger DNA.
Page 1138

Figure 30-5 Semidiscontinuous DNA replication. In DNA

replication, both daughter strands (leading strand red,
lagging strand blue) are synthesized in their 5¢ ® 3¢
directions.
RNA Primers
• Need a 3’-OH group to extend DNA chain.
• Analysis of Okazaki fragments revealed that they have
short (1-60 nt) RNA segments complementary to the
template DNA.
• RNA primers catalyzed by 2 enzymes:
• RNA polymerase, large (459 kD), mediates
transcription, rifampicin sensitive.
• Primase (DnaG), small (60 kD), rifampicin resistant.
• Rifampicin inhibits only leading strand synthesis,
therefore, primase initiates Okazaki fragments.
• Leading strand can be mediated in vitro by both, is
better when both enzymes present.
 Figure 30-7 Priming of DNA synthesis by short RNA
segments.
Page 1139
Figure 11.8 Closeup of a replication fork showing initiation
of the continuous leading strand and the discontinuous,
lagging strand (Okazaki fragments)

• All known DNA polymerases catalyze chain formation in the 5’  3’

direction.
• DNA strands must be copied in both directions!
Figure 11.9a Complete scheme showing sequential steps of
replication process.

1. Helicase (unwinding protein / rep protein) recognizes

and binds origin of replication.
• Catalyzes separation of the two DNA strands by disrupting H-
bonding between base pairs.
• Endothermic reaction is coupled to hydrolysis of ATP.
• DNA gyrase (a topoisomerase) assists in unwinding by inducing
supercoiling.
Figure 11.9b Complete scheme showing sequential steps of
replication process.

2. Single-stranded DNA binding proteins (SSB) bind

exposed strands of DNA.
• Protect it from hydrolytic cleavage of phosphodiester
bonds.
 Figure 11.9c Complete scheme showing sequential steps of
replication process.

3. Primer synthesis: Short complementary stretch of RNA

(4-10 bases) is synthesized by primase enzyme.
• Primer with free 3’-OH is required by DNA pol III to start
2nd strand synthesis.
• RNA primer is later degraded by 5’->3’ exonuclease
action of DNA pol I, RNaseH enzymes.
Figure 11.9d Complete scheme showing sequential steps of
replication process.

4. DNA synthesis by DNA pol III begins, extending leading

and lagging strands.
• DNA synthesis continues until it meets next fragment.
Figure 11.9e Complete scheme showing sequential steps of
replication process.

5. RNA primers are removed by 5’3’ exonuclease action

of DNA polymerase I, small gaps are filled in by DNA
polymerase I.
Figure 11.9f Complete scheme showing sequential steps of replication
process.

6. Final gap between new strands is closed by DNA ligase

enzyme.
• Requires ATP to join 3’ OH on one fragment and 5’
phosphate on second fragment.
Figure 11.10 The DNA ligase-catalyzed reaction to close the
final phosphodiester bond in newly synthesized DNA.

ATP is required as a source of energy for this endergonic

reaction.
DNA replication proteins
Eukaryotic DNA polymerases

• Several eukaryotic DNA polymerases also known:

• a found only in nucleus, requires template and primer.
• b, d, e also found in nucleus
• g found in mitochondria
Table 6–1 Error Rates in DNA synthesis

If an incorrect nucleotide is added to a growing strand, the DNA

polymerase will cleave it from the strand and replace it with the
correct nucleotide before continuing.
DNA Pol I is a proofreading enzyme

• 3 active sites on the same enzyme: DNA polymerase activity, a 3’

 5’ exonuclease and a 5’  3’ exonuclease.
• 5’  3’ exonuclease activity is independent of the 3’  5’
exonuclease and polymerase activity.
• Pol I can be cleaved by trypsin into two fragments: larger C-
terminal fragment is called the Klenow fragment (KF) and
contains the polymerase and 3’  5’ exonuclease activity. The N
terminal fragment contains the 5’  3’ exonuclease activity.
• Pol I is a processive enzyme; it catalyzes a series of successive
polymerization events (20 or more) without releasing the
template.
Exonuclease activity of DNA polymerase I

• (a) 3’5’ exonuclease activity

DNA polymerase I acts as a proofreading and repair enzyme by

catalyzing hydrolytic removal of mismatched bases.
Exonuclease activity of DNA polymerase I

• (b) 5’3’ exonuclease activity

DNA polymerase I acts as a proofreading and repair enzyme by

catalyzing hydrolytic removal of mismatched bases.
 Schematic diagram for the nucleotidyl transferase
mechanism of DNA polymerases.

2 metal ions (Mg2+)

B ligands the
phosphates of dNTP
A bridges Pa with
the primer’s 3’OH for
in-line nucleophilic
attack.
Pol I
• 3’-5’ exonuclease functions in editing DNA.
• Crystal structure shows a 12 nt “template” strand (5’-
TGCCTCGCGGCC-3’) and a 7 nt “primer” strand (3’-
GCGCCGG-5’) that is complementary to the 3’ end of
the template strand and a 2’,3’-epoxy-ATP.
• Forms distorted segment.

O
2’,3’-epoxy-ATP
template

primer
Pol I

• A second primer strand is also present that base pairs

with the 5’-terminal nucleotides but has a single T
overhang on the template strand and a 3 nucleotide
overhang on the primer strand.
• The 3’-terminal nucleotide of the primer strand is
bound at the 3’-5’ exonuclease site (editing complex).
• The 3’ end of the primer strand is in competition to go
between the active site and editing site. If properly
base paired it goes to the active site, otherwise it goes
in the editing site.
 Probable sequence of the double-stranded DNA
seen in the X-ray structure of KF.
Page 1144
Other Pol I activities

• Pol I functions to repair DNA by simultaneously filling

in single-strand gaps formed during endonucleolytic
cleavage of DNA.
• The 5’-3’ exonuclease and polyerase activities can
replace nt on the 5’ side of a single strand nick.
• These reactions move the nick toward the 3’ end of
the DNA strand (nick translation).
• Can be used to create radiolabeled DNA.
Nick translation as catalyzed
by Pol I.
Other Pol I activities

• The 5’-3’ exonuclease activity also removes RNA

primers at the 5’ ends of newly synthesized DNA.
• DNA polymerase fills in gaps.
• Used temperature-sensitive E. coli mutants to
demonstrate the function.
• Essential for survival.
DNA Pol III is the replicase

• Pol III core has multiple subunit composition. The polC

gene encodes the polymerase function.
• Has a 3’ 5’ exonuclease editing function.
• 5’  3’ exonuclease activity can only act on single
stranded DNA.
• Pol III core has processivity for only 10-15 residues.
• Needs the b-subunit in the presence of the g complex for
maximum processivity (>5000 residues).
• b-subunit forms a sliding clamp on the DNA.
Components of E. coli DNA Polymerase III
Holoenzyme.
X-Ray structure of the b subunit of E. coli Pol III
holoenzyme. Ribbon drawing.
The b subunit of E. coli Pol III holoenzyme. Space-filling
model of sliding clamp in hypothetical complex with B-
DNA.
DNA is unwound by Helicases

• Helicases are involved in DNA replication,

recombination, repair and transcription termination,
RNA splicing, and RNA editing.
• Translocate on one strand of double-helical nucleic acid to
unwind the double helix using the energy of hydrolysis of
NTPs.
• Classified by their direction: 5’-3’ and 3’-5’ helicases.
 Unwinding and Binding Proteins of E. coli DNA
Replication.
Page 1146
Helicases

• DnaB-hexameric helicase, 5’-3’ translocation along the

lagging strand, hydrolyzes ATP. Cannot use UTP. No
crystal structure.
• T7 gene 4 helicase/primase-2-tiered hexagonal ring with
N-terminal domains containing the primase activity and C-
termini carry out the helicase activity. Uses dTTP (also
dATP and ADP). Crystal structure known (Hexameric C-
termini similar to DnaB).
Unwinding of DNA by the combined action
of DnaB and SSB proteins.
Electron microscopy–based image reconstruction
of T7 gene 4 helicase/primase.
 X-Ray structure of the helicase domain of T7 gene 4
helicase/primase.
Page 1147
Helicases

• Rep helicase- monomeric enzyme, but active form is a

dimer with 3’-5’ translocation.
• Shows negative cooperativity, one subunit binds to ssDNA
and inhibits the binding of the other subunit to
ssDNA(binds to dsDNA).
• Enzyme operates through “active/rolling” mechanism.
• Unwinds DNA by walking up strand in ATP dependent
manner.
• PriA -is a similar monomeric enzyme with 3’-5’
translocation
Active, rolling mechanism for DNA unwinding by
Rep helicase.
 X-Ray structure of Rep helicase in complex with
dT(pT)15 and ADP.
Open Closed
•Two domains (1 and
2)
•Each domain is in 2
subdomains (1A, 2A-
N-terminal subdomain)
•Change occurs in ssDNA
domain 2B from open
to closed

ADP
 X-Ray structure of the N-terminal 135 residues of E.
coli SSB in complex with dC(pC)34.
ssDNA

•SSB prevent ssDNA from

reannealing to form dsDNA
•Homotetramer
•2 major forms (subscript is # of
nt)
•(SSB)35 in which 2 of of the four
subunits interact with ssDNA
•(SSB)35 shows unlimited
cooperativity
•(SSB)65 in which all 4 of the
subunits interact with ssDNA;
limited cooperativity.
 The reactions catalyzed by E.
coli DNA ligase.

• Can use either NAD+ converted to

NMN+ +AMP or ATP to PPi and AMP.
1. The adenylyl group of NAD+ is
transferred to e-amino group of Lys to
form phosphoamide addict (unusual).
2. Adenylyl group of activated enzyme is
transferred to 5’-phosphoryl terminus of
nick to form adenylylated DNA (AMP
linked to 5’nucleotide via PPi.
3. DNA ligase catalyzes formation of
phosphodiester bond by attack of the
3’-OH group on the 5’-nucleotide.
Primase

• Closely associated with DNA helicase (T7 gene 4

helicase/primasehas both domains)
• E. coli primase (DnaG) forms noncovalent complex
with DnaB
• Primase reverses its direction in order to synthesize
RNA primer in 5’-3’ direction.
• 3 domains: N-terminal Zn2+ binding domain, central
catalytic domain with Mg2+ , C-terminal domain
interacts with DnaB.
X-Ray structure of E. coli primase.