COURSE: BIOCHEMISTRY 301: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare 2019

WEEK 2: DNA
REPLICATION

Structure recap

DNA is polymeric
Single nucleotide monomers are joined together by covalent bonds to form polynucleotide chains of either DNA
or RNA. The DNA strand contains 3’ – 5’ phosphodiester bonds and, since the bond is directional, this leads to a
strand that has direction (or “sense”).

Base pairing
RNA = single stranded (covalent intranucleotide bonding)
DNA = double stranded (interchain bonding)

Complimentary pairing of DNA bases:

A pairs with T, C pairs with G

Look up Chagraff’s Rule and The Watson and Crick DNA Double Helix Model from your BCH201 notes

When Watson and Crick proposed the double helical structure of DNA with its complementary base pairing, they
immediately recognized that base pairing specificity could provide the basis for duplication. "It has not escaped
our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for
the genetic material.“ Nature 171: 737-738 (1953). If the two complementary strands of a double helix were
separated (by breaking the H2 bond), each parental strand could direct the synthesis of a new complementary
strand ie. each parental strand could serve as a template for a new complementary strand.
 COURSE: BIOCHEMISTRY 301: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare 2019

Hyperchromic shift and DNA melting

The double helix can be separated reversibly (DENATURATION) and reformed (RE-ANNEALING)

Temperature at which the DNA strand separates is called the melting temperature (Tm) – this is when 50% of the
DNA in a sample is separated into single strands. The absorbance at 260 nm increases as base pairs are exposed
(hyperchromic shift). Tm of DNA increases with an increase in the GC content of the DNA. This is due to the
fact that there are three hydrogen bonds between guanine and cytosine as opposed to the two hydrogen bonds
between adenine and thymine

DNA REPLICATION
In considering possible mechanism of DNA replication, three different hypothetical modes are apparent:
Conservative (Old-old and new-new), Semi-conservative (2 x old-new) or Dispersive/distributive (Covered in
BCH201, recap).

The Meselson – Stahl Experiment (Covered in BCH201, recap).

 COURSE: BIOCHEMISTRY 301: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare 2019

General Requirements for DNA replication

1. START: Origin of Replication
2. Single-stranded template strand (base pairing)
3. Nucleotides (dNTPs)
4. Metal ions (usually magnesium) - One Mg2+ activates the 3’-OH for nucleophilic attack. Second Mg2+
orientates the triphosphate group of the incoming nucleotide, and electrostatically shields their negative
charges
5. Primer with free 3`-OH
6. Enzymes and associated proteins

Origin of replication (ORI)

This is the position or positions in the genome at which DNA replication starts. The sequences are species
specific but share some features:
- Short consensus sequences
- High AT %, low GC% (easier to separate)

Enzymes and Proteins involved in DNA replication

PRIMASE: Enzyme that synthesizes a short strand of RNA on a DNA template, producing a primer for DNA
synthesis.
HELICASE: Enzyme that is involved in opening the DNA helix into its single strands for DNA replication (ATP
dependent)
DNA POLYMERASES: Enzyme that synthesizes DNA by joining nucleotides together using a DNA template as
a guide
LIGASE: Enzyme that joins the ends of two strands of DNA together with a covalent bond to make a
continuous DNA strand.
TOPOISOMERASE I OR GYRASE: Enzyme that prevents DNA tangling during
replication. TELOMERASE: Enzyme that elongates telomere sequences in DNA

DNA synthesis is SEMI-DISCONTINOUS

Leading strand synthesis is continuous, while the synthesis of the lagging strand is discontinuous.

Leading strand:
1. Helicase unwinds and unzips
2. DNA polymerase III adds complimentary DNA nucleotides

Lagging strand is:

Formed in short segment of 100-200 nucleotides (these are the Okazaki fragments)
Grows in direction of 5’→3’ because DNA polymerase III only works in the 5’→3’
1. Helicase unwinds and unzips the parent DNA
2. RNA primer is formed by RNA nucleotides that are joined together by RNA primase
3. DNA polymerase III bonds DNA nucleotides to the RNA primer
4. DNA polymerase I replaces the RNA primer with DNA nucleotides
5. DNA ligase joins the Okazaki fragments together
 COURSE: BIOCHEMISTRY 301: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare 2019

DNA ligase activity

Why is the lagging strand synthesized discontinuously?

Because the lagging strand needs to be replicated in the opposite direction of the way the replication fork is
proceeding. And DNA polymerase can proceed only 5' to 3'. Thus, short fragments are produced as the replication
fork expands.
Otherwise DNA polymerase would need to wait until the fork reached the end of the molecule to replicate the
lagging strand.

Okazaki fragments
Experimental evidence supporting the hypothesis that DNA replication is a discontinuous process:
1. To distinguish the method of replication used by DNA experimentally, newly replicated areas of
Escherichia coli chromosomes were pulse-labeled, denatured, and extracted the DNA. A large amount
of radioactive short units meant that the replication method was likely discontinuous. The hypothesis
was further supported by the discovery of polynucleotide ligase, an enzyme that links short DNA
strands together.
E. coli were infected with bacteriophage T4 that produce temperature-sensitive polynucleotide ligase. The cells
infected with the T4 phages accumulated a large amount of short, newly synthesized DNA chains when exposed to
high temperatures as predicted in the hypothesis

GLOSSARY OF TERMS
- Replication fork: The junction between the newly separated strands and unreplicated double stranded DNA
- Leading and Lagging strand: Due to the anti-parallel nature of DNA, one strand will be synthesized
continuously towards replication fork and other strand will be synthesized discontinuously away from the
replication fork. The continuously synthesized strand is called the leading strand and the discontinuously
synthesized strand is called the lagging strand.
- Okazaki fragment: A short fragment of DNA formed on the lagging strand during replication is called an
Okazaki fragment. It will be around 100 – 1000 bp in length.
- Processivity: The ability of an enzyme to catalyze many reactions before releasing its substrate is called
processivity. NB concept!
 COURSE: BIOCHEMISTRY 301: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare 2019

DNA REPLICATION IN PROKARYOTES

ORI: Prokaryotes
Prokaryotes possess a circular chromosome , with often a single origin of replication. This is recognised by
protein complexes. In E. coli the minimal recognition site is a 245 bp sequence that contains adjacent AT-rich
region and Four inverted repeat sequences bound by replication proteins containing sequence repeats.

Prokaryotic DNA Replication Proteins

- Primosome: Primase
Helicas
e
- DNA polymerase III holoenzyme
- DNA polymerase I
- Ligase
- Single-strand binding protein (SSB)
- Topoisomerase I or Gyrase

Activities of Prokaryotic Polymerases

Polymerase I
- 5’ -3’ polymerase: DNA synthesis, extension
- 3’ - 5’ exonuclease: Proof-reading
- 5’ - 3’ exonuclease: Removes primers at the beginning and fills in the gaps with polymerase activity
Polymerase II and III
- 5’ -3’ polymerase: DNA synthesis, extension
- 3’ - 5’ exonuclease: Proof-reading

Polymerase I can be divided into two fragments:

Large C-terminal fragment (Klenow fragment)
- Residues 324-928
- Polymerase activity
- 3’ → 5’ exonuclease activity
Smaller N-terminal fragment
- Residues 1-323
- 5’ → 3’ exonuclease activity
 COURSE: BIOCHEMISTRY 301: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare 2019

DNA polymerase III

- The DNA polymerase III holoenzyme is a multisubunit complex, which consists of 17 polypeptides.
- It contains four subassemblies.
- The core polymerase consists of three subunits: the polymerase; the 3'–5' exonuclease and the
stimulator of the 3'–5' exonuclease.
- The subunit responsible for dimerization of the core DNA polymerase.
- The sliding clamp comprises two homodimers of the subunit, which provides the ring structure that is
needed for processivity.

Transcription termination
In prokaryotes this is mediated by:
1. Specific terminator sequences (Ter sites).
2. Proteins called Tus proteins which bind to these sequences and prevent the replication forks from
continuing beyond a particular point (two Tus proteins work in tandem)

Eukaryotes do not appear to have such a mechanism (likely due to the linear nature of the DNA and the
presence of telomeres)

Eukaryotic DNA Replication

Humans: 30 000 – 50 000 ORIs
Found in regions that lack nucleosomes
Sequence Correlates with areas of the genome that are being transcribed (especially transcription start sites in the 5`
region of the genome)
 COURSE: BIOCHEMISTRY 301: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare 2019

Includes T rich and A rich sequences)

Eukaryotes: α , β, γ δ ε polymerases
DNA polymerase α - lagging strand priming / initiator polymerase
DNA polymerase β – base excision repair polymerase
DNA polymerase γ - mitochondrial replicase
DNA polymerase δ - leading strand and lagging strand elongation/main polymerae
DNA polymerase ε - leading and lagging strand polymerase

Other proteins and enzymes

- PCNA protein (proliferating cell nuclear antigen): complexes with DNA pol, this is the clamp for the
DNA polymerase, equivalent to the β sub-unit of prokaryotic DNA polymerase III
- RFC (replication factor C): this is the clamp loader, equivalent to sub-unit γ of prokaryotic DNA
polymerase III
- RPA Replication Protein A – binds single stranded DNA at the opening of the replication fork,
equivalent to SSB (single stranded DNA binding protein) in prokaryotes
RNaseH1 and Flap endonuclease -1 (FEN-1)
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

Features of DNA replication specific to eukaryotes

• In eukaryotes DNA replication is controlled by a system known as the cell cycle.
• Cell growth is continuous but DNA is only replicated once during the cycle.
• Four stages:
- G1 (Gap 1): Growth phase
- S (Synthesis): DNA replication
- G2 (Gap 2): Preparation for mitosis
- M (Mitosis): Separation of duplicated chromosomes
- G0: Specialised quiescent state where cells are metabolically active but do not replicate DNA

Prokaryotic vs eukaryotic DNA replication:

TELOMERES
Telomeres are specialized protein-DNA structures that make up the ends of chromosomes in eukaryotes. They
contain thousands of repeats of G-rich consensus sequence. In mammalian cells, telomere DNA contains
double-stranded tandem repeats of TTAGGG followed by terminal 3’ G-rich single-stranded over- hangs.
Telomere DNA is thought to adopt the T-loop structure, where the telomere end folds back on itself and the 3’
G strand overhang invades into the double-stranded DNA. In humans there are 46 chromosomes and thus 92
telomeres (one at each end).
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

Telomeres contain a multi protein complex

- Mammalian telomeres possess a six protein complex called “SHELTERIN”.
- TRF1 and TRF2 bind to the TTAGGG sequences in the double strand telomeric DNA.
- POT1 binds to the sequences in single strand form
- TIN2 and TPP1 proteins keep TRF1, TRF2 and POP1 together.
- This six protein complex, SHELTERIN prevents the activation of the DNA damage response.
- SHELTERIN is required for the recruitment of telomerase.

Function of telomeres
- They protect the ends of chromosomes.
- They separate one chromosome from another in the DNA sequence
- Without telomeres, the ends of the chromosomes would be "repaired", leading to chromosome
fusion and massive genomic instability (ie. they prevent end-to-end joining)

The End-replication problem

The usual replication machinery provides no way to complete the 5’ ends of daughter DNA strands. In 1972,
James Watson called this the “End-Replication Problem.”The problem arises on the LAGGING strand. As a
result, linear chromosomes shorten by 50 – 100 nucleotides with each cell division. Therefore, cells have a
fixed/finite number of cell divisions and long lived cells require a mechanism to replicate the ends of linear
chromosomes . The problem: DNA polymerase/ligase cannot fill gap at the end of the chromosome after the
RNA primer is removed. If this gap is not filled, chromosomes would become shorter each round of replication.
The solution: Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. Telomerase
(composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and
catalyzes the addition of new repeats. This compensates by lengthening the 5` to 3` strand to provide a primer
binding site that allows the extension of the 3` - 5` strand. Absence or mutation of telomerase activity results in
chromosome shortening and limited cell division. Telomerase is only active in long lived cells such as stem cells
or cancer cells.
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

Telomerase
• Telomerase is an enzyme that preserves the length of telomeres across cell divisions.
• Telomerase is over-expressed in certain cancers. It is also active in germ cells and stem cells, but
lacking from adult cells.
• It is a ribonucleoprotein that carries its own primer, an RNA sequence complimentary to the telomere.
This RNA acts as template for the extension of the lagging strand. The enzyme has reverse transcriptase
activity [RNA-directed DNA polymerase].
• Active telomerase is a complex of:
- 2 molecules Telomerase Reverse Transcriptase (TERT)
- Telomerase RNA (hTR or TERC)
- Dyskerin

Telomeres and cancer

Telomeres were first discovered in cancer cells due to the fact that these cells are often saturated with telomerase
enzyme. As a cell becomes cancerous, it divides more often and its telomeres become very short. If its telomeres
get too short, the cell may die. It can escape this fate by activating either telomerase or an alternative pathway,
resulting in abnormal telomere lengthening & proliferative growth
Therefore, telomerase is over expressed in many cancers cells.

Telomeres and ageing

Healthy human cells are mortal because they can divide only a finite number of times, growing older each time
they divide. It has been proposed that telomere shortening may be a molecular clock mechanism that counts the
number of times a cell has divided and when telomeres are short, cellular senescence (growth arrest) occurs.
Jerry Shay and colleagues found that cellular aging can be bypassed or put on hold by the introduction of the
catalytic component of telomerase. In the laboratory, cells in tissue culture with introduced telomerase have
extended the length of their telomeres. They are able to divide for up to 250 generations past the time they
normally would stop dividing.
But remember: humans age due to factors other than telomere length, eg oxidative stress and mitochondrial
function impairment of cells, increase in risk factors for major diseases increases with age.

THE POLYMERASE CHAIN REACTION (PCR)

Polymerase chain reaction: is a molecular biology process that allows the exponential synthesis of a specific
section of DNA by controlling the process of DNA replication under laboratory conditions. It “mimics DNA
replication in a test-tube.” PCR combines all of the requirements for DNA synthesis in a test-tube and then
induces synthesis of specific DNA by going through a series of cycles in which the temperature is changed to
promote a specific enzyme activity.

Requirements for PCR

1. DNA template: that contains the DNA sequence to be amplified;
2. Forward and reverse primers: that define which section of DNA will be amplified. These primers will
base-pair will a specific sequence on the template DNA;
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

3. Nucleotides: monomers that will be joined into new DNA strand by the polymerase;
4. Polymerase: enzyme that catalyses the formation of phosphodiester bonds between nucleotides;
5. Cofactors: buffers and Mg;
6. Thermocycler: to control the rapid changes in temperature required between the different cycles.

PCR cycling parameters

Three steps
- Denaturation: strand separation by breaking of H bonds at high temperatures
(90–95 °C).
- Annealing: cooling (40 – 60 °C) allows primer annealing. Two primers (forward and reverse) are supplied in
molar excess They bind to the complementary region. As the DNA cools, they wedge between two template
strands. Temperature dependent on primer characteristics eg length, GC content (Tm minus 5 °C).
- Extension: DNA polymerase adds dNTPs to annealed primer in order to synthesize a new strand (at 72 °C). 1
minute required per kb of DNA to be synthesized

Example PCR parameters (simple)

Step Description Temp Time No. of cycles

1 Initial 92 °C 2 min 1
denaturation
2 Denaturation 92 °C 30 sec 25
Annealing 55 °C (Tm of 45 sec
primer 60 °C)
Extension 72 °C 1 min
(1 kb product)
3 Final extension 72 °C 3 min 1

TAQ polymerase
- Derived from Thermophilus aquaticus
- Heat stable DNA polymerase
- Optimum temperature 72 °C
- An initial complication for PCR was that the DNA polymerase used was destroyed by the high heat used
at the start of each replication cycle and had to be replaced. In 1986, Mullis started to use Taq DNA
polymerase to amplify segments of DNA.
- This was what made the difference in that PCR was now more affordable and could be automated.
In addition:
- Generates a single template-independent nucleotide at the 3´ end of each DNA strand (Clark, 1988;
Newton and Graham, 1994). This single-nucleotide overhang is most commonly an A residue which
allows cloning into vectors such as pGEM T Easy which have T overhangs (see later)

How is PCR different to in vivo synthesis?

- In PCR, the DNA to be replicated is separated by heat denaturation. In vivo, DNA is separated by an
ATP-dependent helicase.
- In vivo, DNA primases produce RNA primers in a sequence-independent manner on exposed single-
stranded DNA. 5'->3' exonuclease activity to degrade RNA primers is therefore unnecessary for PCR.
(Before Taq, the primary polymerase used for PCR was the Klenow fragment.)
- DNA replication forks (and the Okazaki fragments associated with lagging strand synthesis) do not occur
in PCR. Why?
- The rate of DNA synthesis is 1 kb/s in E. coli but 1-4 kb/min in a typical PCR reaction. Rates of
misincorporation are higher in PCR reactions using Taq than in typical model organisms (1 in 9000 for
Taq vs. 1 in 100,000 for Pol δ/ε in mammals

PCR TROUBLESHOOTING - See tables on RUConnected slides

 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

An introduction to DNA sequencing

PCR using a mixture of dNTS and ddNTPs. The latter block the formation of the next phosphodiester bond
during DNA synthesis
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

The sequencing process is now automated. Fluorescent dye coupled to reaction allows visualization of di-deoxy
termination events by means of a laser that detects the colored product

DNA DAMAGE AND REPAIR

DNA damage refers to a change in the chemical structure of DNA eg. A break in the DNA strand, a change in the
structure of a nitrogenous base, a missing nitrogenous base from a nucleotide. The vast majority of DNA damage
affects the primary structure of the double helix
(vs mutation which is a heritable change in the sequence of bases and cannot be repaired)

Causes of DNA damage

1. Chemical agents acting on the DNA (carcinogens)
2. UV light imparting energy into DNA molecule
3. Spontaneous changes to the DNA

Common types of DNA damage

Deaminations: Three of the four bases normally present in DNA (cytosine, adenine, and guanine) contain amino
group (NH2). The loss of the amino group (deamination) can occur spontaneously and result in the conversion of
the affected bases to uracil, hypoxanthine, and xanthine, respectively.
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

Pyrimidine dimerization: this is not a rare event: every second you are in the sun, 50 to 100 of these dimers are
formed in each skin cell Common causes of DNA damage

Common causes of DNA damage

1. Intercalating agents: Substances whose dimensions are roughly the same as those of a purine-
pyrimidine pair. In aqueous solutions, these substances form stacked arrays, and are also able to stack
with a base-pair by insertion between two base-pairs. This may result in a frameshift mutation.
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

2. Carcinogens that mutate by base alkylation: alkylating agents are electrophilic compounds with
affinity for nucleophilic centers in organic macromolecules. These include a wide variety of chemicals,
many of which are proven or suspected carcinogens (such as nitrous acid, hydroxylamine, and
ethylmethane sulfonate, EMS). They add an alkyl group to a hydrogen-bonding oxygen of G or T,
resulting in G-T mispairing

MUTATION
Mutations are heritable permanent changes in a genomic sequence

Opportunity for errors during DNA replication

Fidelity of DNA polymerases

- All DNA polymerases have an error rate. This means that during the replication of DNA they will insert
an incorrect base at a particular frequency. This is known as the “fidelity” of the enzyme.
- Low fidelity = high error
rate High fidelity = low
error rate

The consequences of mutation

Mutations in the genetic code can:
- Have no effect
- Alter the product of a gene (protein or RNA)
- Prevent the gene from functioning properly or completely
About 70 percent of these mutations (within a gene) having damaging effects
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

Classification of mutations
- By effect on structure
 Small-scale mutations
 Large-scale mutations in chromosomal structure

- By effect on function
 Loss-of-function mutations
 Gain-of-function mutations
 Lethal mutations
 A back mutation or reversion
- By effect on fitness
 A harmful mutation
 A beneficial mutation
- By impact on protein sequence
 A frameshift mutation
 A nonsense mutation
 A missense mutations
 A neutral mutation

Mutations that alter protein sequence

Frameshift mutation: addition or deletion of one base-pair result in a shift of reading frame and alter amino acid
sequence


 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

DNA REPAIR
Damaged DNA must be repaired. If the damage is passed on to subsequent generations, then we use the
evolutionary term - mutation. It must take place in the germ cells. If damage is to somatic cells (all other cells of
the body except germ cells) then just that one individual is affected.

Mechanisms of DNA repair

1. Mismatch repair system (MMR)
2. Nucleotide excision repair (NER)
3. Base excision repair (BER)

1 Mismatch repair
To repair mismatched bases, the system has to know which base is the correct one. In E. coli, this is achieved by a
special methylase called the "Dam methylase", which can methylate all adenines that occur within (5')GATC
sequences. Immediately after DNA replication, the template strand has been methylated, but the newly
synthesized strand is not methylated yet. Thus, the template strand and the new strand can be distinguished.
The repairing process begins with the protein MutS which binds to mismatched base pairs. Then, MutL is
recruited to the complex and activates MutH which binds to GATC sequences. Activation of MutH cleaves the
unmethylated strand at the GATC site. Subsequently, the segment from the cleavage site to the mismatch is
removed by exonuclease The gap is filled by DNA polymerase III and DNA ligase.
The distance between the GATC site and the mismatch could be as long as 1,000 base pairs. Therefore, mismatch
repair is very expensive and inefficient.
.

In mammals the newly synthesised strand is preferentially nicked and can be distinguished in this manner from the
parental strand.
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

2 Excision Repair
The most ubiquitous repair mechanism, which can deal with a large variety of structural defects in DNA.

A) Nucleotide excision repair

NER is a mechanism by which the cell can prevent unwanted mutations by removing UV-induced DNA
damage (mostly thymine dimers and 6-4-photoproducts). Severe human diseases result from genetic mutations of

NER proteins eg Xeroderma pigmentosum, Cockayne's syndrome.

NER enzymes recognize bulky distortions in the shape of the DNA double helix. Recognition of these distortions
leads to the removal of a short ss DNA segment that includes the lesion, creating a single-strand gap in the DNA,
which is subsequently filled in by DNA polymerase, using the undamaged strand as a template.

In E. coli, proteins UvrA, UvrB, and UvrC are involved in removing the damaged nucleotides. First, a UvrA-
UvrB complex scans the DNA, with the UvrA subunit recognizing distortions in the helix. When the complex
recognizes such a distortion, UvrA exits and UvrB melts the base pairs between the two DNA strands. UvrB then
recruits UvrC, which incises the DNA 8 nucleotides away from the distortion on the 5’ end and 4 nucleotides
away on the 3’ end, creating a 12-nucleotide long single-stranded DNA that is excised with the help of the DNA
helicase II. The resultant gap is then filled in using DNA polymerase I and DNA ligase. The basic excision process
is very similar in higher cells, but these cells usually involve many more proteins

Global genomic NER

Global genomic NER repairs damage in both transcribed and untranscribed DNA strands in active and inactive
genes throughout the genome. This pathway employs several 'damage sensing' proteins including the DNA-
damage binding (DDB) and XPC-Rad23B complexes that constantly scan the genome and recognize helix
distortions.

Transcription coupled repair

When RNA polymerase II is transcribing a gene and encounters a helix distorting thymine dimer, for example,
it cannot continue transcription and stalls, recruiting nucleotide excision repair proteins to itself. This has two
important consequences:
1) the RNA polymerase itself becomes a tool for recognizing DNA damage
2) genes that are being actively transcribed are given more attention by the repair mechanisms.
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

A) Base excision
DNA's bases may be modified by deamination or alkylation. BER involves flipping the mutated base out of the
DNA helix and repairing the base alone. There are two main enzymes used: DNA glycosylases and AP
endonucleases. BER is initiated by DNA glycosylase with the recognition of either a specific type of damaged
DNA structure or an inappropriate base, such as uracil. The DNA glycosylase is used to break the β-N glycosidic
bond between the sugar and base in the nucleotide to create an apurinic/apyrimidinic (AP) site. AP endonuclease
recognizes this site and nicks the damaged DNA on the 5' side (upstream) of the AP site creating a free 3'-OH.

DNA polymerase, Pol I, extends the DNA from the free 3'-OH using its exonuclease activity to replace the
nucleotide of the damaged base, as well as a few downstream, followed by sealing of the new DNA strand by
DNA ligase. Usually, BER is divided into short-patch repair (where a single nucleotide is replaced) or long-patch
repair (where 2-10 nucleotides are replaced).

Base excision repair in E. coli

Why mutations are beneficial

If we did not have mutation then we would all be the same!
Any changes in the environment would be deleterious to all members of the population equally
(evolutionary advantage of mutations)
 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 LECTURER: Dr Jo-Anne de la Mare

 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 YEAR: 2018 COORDINATOR: Dr Jo-Anne de la Mare

BCH301 DNA replication J de la Mare

 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 YEAR: 2018 COORDINATOR: Dr Jo-Anne de la Mare

BCH301 DNA replication J de la Mare

 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 YEAR: 2018 COORDINATOR: Dr Jo-Anne de la Mare

BCH301 DNA replication J de la Mare

 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 YEAR: 2018 COORDINATOR: Dr Jo-Anne de la Mare

BCH301 DNA replication J de la Mare

 COURSE: BIOCHEMISTRY 3: Information flow and recombinant DNA techniques

COURSE: Biochemistry 301 YEAR: 2018 COORDINATOR: Dr Jo-Anne de la Mare

BCH301 DNA replication J de la Mare