Chapter 22

DNA Replication

Figure 11.7

Base pairs
proposed by
Watson and
Crick

Covalent and noncovalent interaction

energies

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Figure 11.7

A-form double helix

The A-form of the DNA
double helix. The pitch of the
A-form helix is 2.46; thus the
A-form is a shorter, wider
structure than the B-form.
One turn in A-form DNA
requires 11 bp to complete.
Twelve base pairs are shown
here.

A-DNA in Biology
Forms in dehydrated random-sequence DNA
Stronger tendency for certain sequences e.g.
alternating GC base pairs, high GC content
e.g. binding site for the transcription factor Sp1
Only structure formed by dsRNA
RNA-DNA hybrids formed during replication,
transcription

Figure 29-10b
Nucleotide sugar
conformations. (b) The C2-endo conformation,
which occurs in B-DNA.

Voet & Voet, Biochemistry

Figure 29-10a
Nucleotide sugar
conformations. (a) The C3-endo conformation (on
the same side of the sugar ring as C5), which
occurs in A-RNA and RNA-11.

Voet & Voet, Biochemistry

Comparison of A, B, Z DNA
See table
A: right-handed, short and broad, 2.3 rise, 11 bp
per turn
B: right-handed, longer, thinner, 3.32 rise, 10 bp
per turn
Z: left-handed, longest, thinnest, 3.8 rise, 12 bp
per turn

Comparison of A, B, Z DNA

DNA may adopt a variety of

structures
Quadruplex various types
i-motif
Triple helices (triplexes)
DNA enzymes

DNA Quadruplex Structures

G-quadruplexes are cyclic
arrays of four G residues
united through Hoogsteen
base pairing.

G-quadruplex showing the

cyclic array of guanines
linked by Hoogsteen
hydrogen bonds.

DNA Quadruplexes
Can Adopt Various Topologies
Tetramolecular,
Bimolecular

Unimolecular

DNA Quadruplex Structures

Structure of d(G4T4G4)2K+
solved by X-ray
crystallography. Two
d(G4T4G4)2 strands come
together as hairpins to form
a G-quadruplex. The
backbones of the two
strands are traced in violet.

Roles of G Quadruplexes in Biochemistry

Requirement: 5-G3N1-7G3N1-7G3N1-7G3-3
Functions:
Transcriptional and translational Regulation

Components of the telomere

Origins of replication

Viruses regulate viral infection cycle; immune evasion, chronic

infection
Epstein-Barr virus G4 elements regulate translation, assist viral
latency
HPV regulatory G4 elements help immune evasion, maintain chronic
infections
HIV G4 elements linked to reverse transcription, transcriptional
regulation

Information
Transfer in Cells

The fundamental process

of information transfer in
cells.

DNA replication is semiconservative one of the

two original strands is conserved in each
progeny molecule
DNA replication is bidirectional it proceeds in
both directions from the starting point
Replication requires unwinding of the DNA helix
DNA replication is semidiscontinuous - the
lagging strand is formed from Okazaki
fragments, which are joined to form the final
product

Bidirectional Replication

Comparison of labeling
during unidirectional
versus bidirectional
replication.

An autoradiogram of E. coli chromosome replication.

10

11

Three Stages of DNA Replication

Initiation
Correct assembly of replication proteins at the
starting point (oriC); separate dsDNA
Elongation
Semiconservative DNA replication catalyzed by
the replication machinery
Termination
Arrival at protein bound to termination
sequence causes disassembly of machinery

Origin of replication in E. coli

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13

SSB
Single-stranded binding
protein
Protects single-stranded
DNA
- from re-annealing
- from nucleases
- from secondary structure
formation
Tetramer; can wrap DNA in
dierent modes

Assembly of prepriming complex

DnaA binds to oriC, assembling the DnaA
oligomer and wrapping DNA around it
ssDNA is exposed and the helicase DnaB is loaded
SSB binds Prepriming complex

Primase adds RNA primer
Polymerase holoenzyme assembles
DnaA disassembles (ATP hydrolyis required)

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Priming

Berg, Tymoczko, Gatto & Stryer, 2012

DNA polymerase reaction

DNA polymerase
catalyzes the
nucleophilic attack by the
3-OH at the primer
terminus upon the phosphate of the
incoming dNTP that is
based paired with the
template
This forms a new
phosphodiester bond
and releases
pyrophosphate

15

DNA polymerase
Discovered 1950s Arthur Kornberg
109 kDa,
Reaction: (DNA)n + dNTP (DNA)n+1 + PPi

Requirements:

1. Requires all four dNTPs and Mg2+
2. New DNA strand is assembled on the
complementary template strand

DNA polymerase

Requirements:

3. Requires a primer
-with free 3 OH group
4. Many polymerases have 3-5 exonuclease
activity (proofreading)

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A Mechanism For All Polymerases

Thomas Steitz has
suggested that all
polymerases use a twometal mechanism. One
metal (A) lowers the
proton affinity of the 3-O
of the growing chain,
promoting nucleophilic
attack on the -P of the
incoming nucleotide.
The second metal (B)
assists departure of the
product PPi.

STRUCTURE OF KLENOW FRAGMENT

Derived by subtilisin
cleavage of DNA
polymerase I
Retains polymerase and 3,
5-exonuclease activity
Does not have 5,3exonuclease activity

Berg, Tymoczko, Gatto & Stryer, 2012

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The exonuclease removes

nucleotides from the 3-end of the
growing DNA chain. The newly
synthesized strand is in purple.

Fidelity
DNA polymerase activity: 1 in 103

With proofreading: 1 in 106

With mismatch repair: 1 in 109

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3'-Exonuclease Activity of Pol I

Removes Nucleotides From the 3'-End
of the Chain
Why does Pol I have exonuclease activity?
The 3'-5' exonuclease activity serves a
proofreading function
It removes incorrectly matched bases, so that the
polymerase can try again
The newly-formed strand oscillates between the
polymerase and 3'-exonuclease sites, adding a
base and then checking it

Properties of DNA Polymerase I

Replication occurs 5' to 3'
Nucleotides are added at the 3'-end of the strand
Pol I catalyzes about 20 cycles of polymerization
before the new strand dissociates from template
20 cycles constitutes moderate "processivity"
Pol I from E. coli is a 928-amino acid (109 kD)
monomer
In addition to 5'-3' polymerase, it also has 3'-5'
exonuclease and 5'-3' exonuclease activities

19

Processivity
DNA polymerase III is a processive enzyme

Processivity ability of an enzyme to catalyze
many consecutive reactions without releasing its
substrate
Processivity conferred by sliding clamp formed by
2

Distributive enzyme releases a polymeric
substrate between successive catalytic steps

DNA Polymerase III

The polymerase that carries out

replication in E. coli
At least 10 different subunits
"Core" enzyme has three subunits - , , and
Alpha subunit is polymerase
Epsilon subunit is 3'-exonuclease
Theta subunit is involved in holoenzyme assembly
and -subunit stabilization
The subunit dimer sliding clamp forms a ring
around DNA
Enormous processivity - 5 million bases!

20

DNA Pol I was discovered in 1957 by Arthur Kornberg and his

colleagues. Pol I and Pol II are involved in DNA repair. Pol III
is the enzyme responsible for replication of the E. coli
chromosome.

Sliding Clamp Holds Pol III on DNA

(a) Ribbon diagram of the -subunit dimer of
the DNA polymerase III holoenzyme on BDNA, viewed down the axis of the DNA.
One monomer of the -subunit dimer is blue
and the other yellow. (b) Space-filling model
of the same structure. The hole formed by
the -subunits is large enough to easily
accommodate DNA (diameter approximately
2.5 nm) with no steric repulsion. The rest of
Pol III associates with this sliding clamp to
form the replicative polymerase.

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Scheme of action of the E. coli clamp loader

Exchange of ADP for ATP increases affinity of the
loader for the clamp
Upon binding, the loader opens the clamp
Binding of the template-primer DNA leads to ATP
hydrolysis, closure of the clamp and release of the
loader

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22

DNA Polymerase III

The Pol III holoenzyme consists of 17 subunits
()222

This is the form that carries out replication

The -subunit is responsible for assembly of the
DNA polymerase III holoenzyme complex
The -complex of the holoenzyme acts as a clamp
loader by catalyzing the ATP-dependent transfer
of a pair of -subunits to the DNA template
Each -subunit dimer forms a closed ring around a
DNA strand and acts as a tight clamp that can
slide along DNA during replication

The Composition of E. coli Pol III

23

One strand synthesized as fragments

The Semidiscontinuous
Model for DNA Replication
(a) Leading and lagging strand synthesis.

Newly
synthesized
DNA is red.

(b) The action of DNA polymerase.

24

Priming

Berg, Tymoczko, Gatto & Stryer, 2012

25

Primase
Specialized DNA-dependent RNA polymerase
Primer is a short RNA (~ 5 nt)
Allows DNA polymerase to start synthesis
Later removed by polymerase 5-3 exonuclease
activity (nick translation)

Helicase
Molecular motor proteins
Hexamers
Use ATP hydrolysis to power separation of the
duplex

26

Supercoils Are One Kind of Structural

Complexity in DNA
Double-stranded circular DNA forms supercoils, if the
strands are underwound, or overwound.

27

28

29

How Does the 5'-Exonuclease Activity

of Pol I Accomplish Nick Translation?
5'-exonuclease activity, working together with the
polymerase, accomplishes "nick translation"
Hans Klenow used either subtilisin or trypsin to
cleave between residues 323 and 324,
separating 5'-exonuclease (on residues 1-323)
and the other two activities (on residues
324-928, the so-called "Klenow fragment)
This 5'-exonuclease activity plays an important
role in primer removal during DNA replication

https://www.youtube.com/watch?
v=yqESR7E4b_8

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Termination of Replication
Termination occurs at ter region of the
chromosome
Ter region is rich in G,T
Tus (terminator utiliation substance) is a protein
that binds to the ter region.
This protein prevents the replication fork from
passing by inhibiting helicase activity.

Tus protein

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Decatenation
Topoisomerase IV
decatenates the two
new, linked circular
chromosomes

Transcription
Synthesis of RNA in cells
Template-dependent synthesis of
polyribonucleotides
Similar to DNA replication: how?
Use of NTP substrates
Template-directed growth of nucleic acid chain
Chain grows in 5-3 direction

32

Transcription
Template-dependent synthesis of
polyribonucleotides
Dierences
Only one DNA strand transcribed
Small fraction of DNA is transcribed.

Important: how does cell select genes for
transcription?

Similarities to DNA replication:

Template-directed synthesis, only in 5 3'
direction

Similar mechanism

Pyrophosphate hydrolysis

NTP substrates; ribonucleotides

33

Mechanism of chain elongation by RNA polymerase

Strand growth
5

RNA product

RNA product

Modified from Tymoczko, Berg and Stryer, Biochemistry, 2nd ed., W.H. Freeman, 2013, Fig. 36.3

Differences with respect to DNA replication:

Not all DNA is transcribed

Only one strand is transcribed

Initiation and termination mechanism used to
select genes for transcription

Primer not required

34

Conventions Used in Expressing the Sequences

of Nucleic Acids and Proteins

Conventions Used in Expressing the Sequences

of Nucleic Acids and Proteins
Certain conventions are used in describing information
transfer from DNA to protein:
The strand of duplex DNA that is read by RNA
polymerase is termed the template stand
The strand not read is the nontemplate strand
The template is read by the RNA polymerase moving
3'-5' along the template, so the RNA product, the
transcript, grows in the 5'-3' direction
By convention, when the order of nucleotides in DNA
is shown, it is the 5'-3' sequence of nucleotides in
the nontemplate strand

35

All Cells Contain Three Major Classes of

RNA mRNA, rRNA, and tRNA
All three forms participate in protein synthesis
All RNAs are synthesized from DNA templates by
DNA-dependent RNA polymerases
This process is called transcription
Only mRNAs direct the synthesis of proteins
Transcription is tightly regulated in all cells
Only 3% of genes in a typical eukaryotic cell are
undergoing transcription at any given moment
The metabolic conditions and growth status of the
cell dictate which gene products are needed at
any moment

36

rRNA Structural, functional form of the

ribosome
Characteristic secondary structure
Sedimentation coecients that indicate size
2 subunits: E. coli 30S, 50S
Eukaryotic: 40S, 60S
80% total cellular RNA;
Contains unusual nucleotides

Ribosomal RNA Provides

the Structural and
Functional Foundation for
Ribosomes
Figure 10.21 Ribosomal RNA
has a complex secondary
structure due to many
intrastrand H bonds. The gray
line here traces a
polynucleotide chain consisting
of more than 1000 nucleotides.

37

Ribosomal RNA

Figure 10.22 The organization and composition of ribosomes.

Ribosomal RNA Provides the Structural

and Functional Foundation for
Ribosomes

Figure 10.23 Unusual bases in RNA.

38

tRNA: Carry amino acids to the ribosome

Unique secondary structure
73-94 residues
Unusual modications: methylation, other
-CCA-3 at 3-terminus
Amino acid aoached as ester to the free 3-OH
Aminoacyl tRNAs are the substrates of protein
synthesis

Transfer RNAs Carry

Amino Acids to
Ribosomes for Use in
Protein Synthesis

Figure 10.24 Transfer RNA also

has a complex secondary
structure due to many intrastrand
hydrogen bonds.

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The Process of Transcription Has Four Stages

Transcription can be divided into four stages:

1) Binding of RNA polymerase holoenzyme to
template DNA at promoter sites
2) Initiation of polymerization
3) Chain elongation
4) Chain termination

Sigma Subunits of Prokaryotic RNA

Polymerases Identify Transcription Start Sites
Transcription is initiated in prokaryotes by RNA polymerase
holoenzyme, with the subunit composition 2'
Binding of the subunit allows the polymerase to recognize
different DNA sequences that act as promoters
Promoters are nucleotide sequences that identify the location
of transcription start sites, where transcription begins
Without bound, the core polymerase can transcribe DNA into
RNA, but cannot initiate transcription

40

Structure of the Core RNA Polymerase from

Thermus thermophilus
The template
DNA strand is
green, the
nontemplate
strand is blue,
and the RNA
transcript is hot
pink. The 2
chains are
orange, the
chain is cyan,
the ' chain is
yellow.

E. coli RNA polymerase

41

Figure 29.3 Sequence

of events in the initiation
and elongation phases
of transcription as it
occurs in prokaryotes.
Nucleotides in this
region are numbered
with reference to the
base at the transcription
start site, which is
designated +1.

Prokaryotic Initiation and Elongation

Figure 29.3 Sequence of events in the initiation and

elongation phases of transcription as it occurs in prokaryotes.

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Prokaryotic Initiation and Elongation

Figure 29.3 continued: Sequence of events in the initiation and

elongation phases of transcription as it occurs in prokaryotes.

Binding of Polymerase to Template DNA

Polymerase binds nonspecifically to DNA with low
affinity and migrates along it, looking for promoter
Sigma subunit recognizes promoter sequence
RNA polymerase holoenzyme and promoter form a
closed promoter complex (in which the DNA is
not unwound)
Polymerase then unwinds about 12 pairs to form
"open promoter complex
RNA polymerase binding protects a nucleotide
sequence spanning the region from -70 to +20,
where +1 is defined as the transcription start site

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Properties of Prokaryotic Promoters

Promoters recognized by the factor typically consist of a
40 bp region on the 5'-side of the transcription start site
Within the promoter are two consensus sequence
elements:
The Pribnow box near -10, with consensus TATAAT
The -35 region, with consensus TTGACA - subunit
binds here. The more the -35 region sequence
corresponds to the consensus sequence, the better the
subunit binds, and the greater is the efficiency of gene
transcription

44