Chapter 5: DNA Replication, Repair,
and Recombination
Goals
• Illustrate how structure of DNA affects its
function
• Describe enzymes involved in replication
• Summarize their functions
• Explain how DNA is repaired and why it
needs to be repaired
DNA Structure (A Review)
• DNA consists of two
strands
– Each strand is a polymer
of nucleotides
– Strand has orientation
due to nucleotide
structure: 5’ and 3’ ends
– The two strands are
antiparallel
DNA Function (A Review)
• DNA function is information storage
• Sequence of strand stores info
– Genes are copied into RNA (transcription)
– “Control elements” regulate protein interactions
with DNA
• DNA passed on to descendant cells
– Accurate copying
– Repair of any damage to avoid changes
– Accurate subdivision
Fig. 2.11
5’ end
3’ end
Fig. 2.15
Functions Determine Form
• Double strands of DNA allow “easy”
replication
– Rules for obligate pairing
– Each strand acts as template for the other
• Multiple proteins involved
– Act in concert
– Act as complexes
– Recognize DNA by shape of bases
Paired DNA Strands
Replication Overview
• Replication complex binds to replication
“origin”
• Double-stranded DNA is “melted”
• Each strand is used as a template for DNA
synthesis
Mechanism of DNA Replication
General Features of DNA Replication
•
•
•
•
Semiconservative
Complementary Base Pairing
DNA Replication Fork is Assymetrical
Replication occurs in 5’
3’ Direction
Semi-conservative Replication
Alternative models of DNA replication (Fig 3.1):
Semi-conservative Replication
Equilibrium density gradient centrifugation (Box 3.1)
DNA Replication
1955: Arthur Kornberg
Worked with E. coli.
Discovered the mechanisms of DNA synthesis.
Four components are required:
1.
dNTPs: dATP, dTTP, dGTP, dCTP
(deoxyribonucleoside 5’-triphosphates)
(sugar-base + 3 phosphates)
2.
DNA template
3.
DNA polymerase I (formerly the Kornberg enzyme)
(DNA polymerase II & III discovered soon after)
4.
Mg
2+
(optimizes DNA polymerase activity)
1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)
DNA Replication
DNA elongation (Fig. 3.4a):
Tools of Replication
• Enzymes are the tools of replication:
• DNA Polymerase - Matches the correct
nucleotides then joins adjacent
nucleotides to each other
• Primase - Provides an RNA primer to
start polymerization
• Ligase - Joins adjacent DNA strands
together (fixes “nicks”)
More Tools of Replication
• Helicase - Unwinds the DNA and melts it
• Single Strand Binding Proteins - Keep the
DNA single stranded after it has been melted
by helicase
• Gyrase - A topisomerase that Relieves
torsional strain in the DNA molecule
• Telomerase - Finishes off the ends of DNA
strands
Initiation of replication, major elements:

Segments of single-stranded DNA are called template strands.

Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.

Initiator proteins and DNA helicase binds to the DNA at the
replication fork and untwist the DNA using energy derived from
ATP (adenosine triphosphate).
(Hydrolysis of ATP causes a shape change in DNA helicase)

DNA primase next binds to helicase producing a complex called
a primosome (primase is required for synthesis),

Primase synthesizes a short RNA primer of 10-12 nucleotides,
to which DNA polymerase III adds nucleotides.

Polymerase III adds nucleotides 5’ to 3’ on both strands
beginning at the RNA primer.

The RNA primer is later removed and replaced with DNA by
polymerase I, and the gap is sealed with DNA ligase.

Single-stranded DNA-binding (SSB) proteins (>200) stabilize
the single-stranded template DNA during the process.
Model of replication in E. coli (Fig. 3.5)
In the Beginning…
• Problems due to structure
– DNA is double-stranded
– Template must be single-stranded
– Strands must be separated
• Separation is difficult due to structure
– Melting strands causes tension elsewhere
– If unrelieved tension can snap DNA strand
Topoisomerase (Gyrase)
•
•
•
•
Relieves stress caused by melting DNA
Cleaves DNA and spins around itself to unwind helix
Type I cleaves one strand, type II cleaves both
Reseals DNA strands after relaxation achieved
DNA Replication
DNA Helicase
• Hydrolyze ATP when bound
to ssDNA and opens up
helix as it moves along
DNA
• Moves 1000 bp/sec
• 2 helicases: one on leading
and one on lagging strand
• SSB proteins aid helicase by
destabilizing unwound ss
conformation
Single Strand Binding Protein
• Binds to DNA with no sequence preference
• Binds tighter to single strand than double
• Keeps separated strands from rejoining
DNA Replication
SSB proteins help DNA helicase destabilizing ssDNA
Primase
• Creates a primer for
DNA polymerase
• Template-dependent
• An RNA polymerase
• Active briefly at
beginning of strand
synthesis
DNA Replication
DNA Primase
• uses rNTPs to synthesize short primers on lagging Strand
• Primers ~10 nucleotides long and spaced ~100-200 bp
• DNA repair system removes RNA primer; replaces it w/DNA
• DNA ligase joins Okazaki fragments
Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:
5’
SSB Proteins
Okazaki Fragments
ATP
1
Polymerase III
2
3
Lagging strand
Helicase
+
Initiator Proteins
3’
primase
Polymerase III
5’
RNA Primer
3’

base pairs
5’
RNA primer replaced by polymerase I
& gap is sealed by ligase
3’
Leading strand
DNA Polymerase
• Enzyme that synthesizes
a DNA strand
• Uses existing strand as
template
• Requires a free “3’ end”
to add new nucleotides
• Has several catalytic
functions
• Several forms exist
Eukaryotic DNA Polymerases
Enzyme
Location
Function
• Pol  (alpha)
Nucleus
DNA replication
– includes RNA primase activity, starts DNA strand
• Pol  (gamma)
Nucleus
DNA replication
– replaces Pol  to extend DNA strand, proofreads
• Pol  (epsilon)
Nucleus
DNA replication
– similar to Pol , shown to be required by yeast mutants
• Pol  (beta)
• Pol  (zeta)
• Pol  (gamma)
Nucleus
Nucleus
Mitochondria
DNA repair
DNA repair
DNA replication
Maintenance of DNA Sequences
DNA Polymerase as Self Correcting Enzyme
• Correct nucleotide has greater affinity for moving polymerase than incorrect
nucleotide
• Exonucleolytic proofreading of DNA polymerase
– DNA molecules w/ mismatched 3’ OH end are not effective templates; polymerase cannot
extend when 3’ OH is not base paired
– DNA polymerase has separate catalytic site that removes unpaired residues at terminus
Maintenance of DNA Sequences
High Fidelity DNA Replication
•
•
Error rate= 1 mistake/109 nucleotides
Afforded by complementary base pairing and proof-reading capability of DNA
polymerase
DNA Replication
DNA Polymerase held to DNA by clamp regulatory protein
•
•
•
•
Clamp protein releases DNA poly when runs into dsDN
Forms ring around DNA helix
Assembly of clamp around DNA requires ATP hydrolysis
Remains on leading strand for long time; only on lagging strand for short time when it
reaches 5’ end of proceeding Okazaki fragments
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond (Fig. 3.7)
DNA Replication
Okazaki Fragments
• RNA that primed synthesis of 5’
end removed
• Gap filled by DNA repair enzymes
• Ligase links fragments together
Extension - Okazaki Fragments
5’
3’
Okazaki Fragment
DNA
Pol.
3’
5’
RNA Primer
DNA Polymerase has 5’ to 3’ exonuclease activity.
When it sees an RNA/DNA hybrid, it chops out the
RNA and some DNA in the 5’ to 3’ direction.
5’
3’
DNA
Pol.
RNA and DNA Fragments
3’
5’
RNA Primer
DNA Polymerase falls off leaving a nick.
5’
3’
Ligase
Nick
3’
5’
RNA Primer
The nick is removed when DNA ligase joins (ligates) the
DNA fragments.
DNA Replication
Initiation and Completion of DNA Replication in Chromosomes
Bacteria
►Single Ori
►Initiation or replication highly regulated
►Once initiated replication forks move at
~400-500 bp/sec
►Replicate 4.6 x 106 bp in ~40 minutes
DNA Replication
Initiation and Completion of DNA Replication in Chromosomes
DNA Replication Begins at Origins of Replication
►Positions at which DNA helix first opened
►In simple cells ori defined DNA sequence 100-200 bp
►Sequence attracts initiator proteins
►Typically rich in AT base pairs
DNA Replication
Initiation and Completion of DNA Replication in Chromosomes
Eukaryotic Chromosomes Have Multiple Origins of Replication
►Relication forks travel at ~50 bp/sec
►Ea chromosome contains ~150 million base pairs
►Replication origins activate in clusters or replication units of 20-80 ori’s
►Individual ori’s spaced at intervals of 30,000-300,000 bp
DNA Replication
Initiation and Completion of DNA Replication in Chromosomes
Mammalian DNA Sequences that Specify Initiation of Replication
►1000’s bp in length
►Can function when placed in regions where chromo not too condensed
►Human ORC required for replication initiation also bind Cdc6 and Mcm proteins
►Binding sites for ORC proteins less specific
•
Each eukaryotic chromosome is one linear DNA double helix
•
Average ~108 base pairs long
•
With a replication rate of 2 kb/minute, replicating one human
chromosome would require ~35 days.
•
Solution ---> DNA replication initiates at many different sites
simultaneously.
Rates are cell
specific!
Fig. 3.17
What about the ends (or telomeres) of linear chromosomes?
Big problem---If this gap is not filled, chromosomes would become
shorter each round of replication!
Solution:
1.
Most eukaryotes have tandemly repeated sequences at the ends
of their chromosomes.
2.
Telomerase (contains protein and RNA complementary to the
telomere repeat) binds to the terminal telomere repeat and
catalyzes the addition of of new repeats.
3.
Compensates by lengthening the chromosome.
4.
Absence or mutation of telomerase activity can result in
chromosome shortening and limited cell division.
The Eukaryotic Problem of Telomere Replication
RNA primer near
end of the
chromosome on
lagging strand
can’t be replaced
with DNA since
DNA polymerase
must add to a
primer sequence.
Different types of Nucleotide Polymerases
1) DNA polymerase
Uses a DNA template to synthesize a DNA strand
2) RNA polymerase
Uses a DNA template to synthesize an RNA strand
(= transcription)
3) Reverse transcriptase
Uses an RNA template to synthesize a DNA strand
Found in many viruses
Telomerase is a specialized reverse transcriptase
Telomerase
Two components of the human telomerase:
– the human RNA subunit(hTR)
5’-CUAACCCUAAC-3’
–The human telomerase
reverse transcriptase(hTERT)
• Function:
Telomerase
– Specialized reverse transcriptase
– Prevents “shortening ends problem” problem by
adding telomeres to the end
– Copies only a small segment of RNA that it carries
by itself
– Requires a 3’ end as a primer
– Synthesis proceeds in 5’ – 3’ direction
– Synthesizes one repeat then repositions itself
– When active provides cell immortality
Telomerase is composed of both RNA and protein
What are telomeres?
• Telomeres are…
– Repetitive DNA sequences at the ends of all
human chromosomes
– They contain thousands of repeats of the sixnucleotide sequence, TTAGGG
– In humans there are 46 chromosomes and thus
92 telomeres (one at each end)
Telomeres
Repeated G rich sequence on one strand
in humans: (TTAGGG)n
Repeats can be several thousand basepairs long. In humans,
telomeric repeats average 5-15 kilobases
Telomere specific proteins, eg. TRF1 & TRF2 bind to
the repeat sequence and protect the ends
Without these proteins, telomeres are acted upon by DNA
repair pathways leading to chromosomal fusions
What do telomeres do?
• They protect the 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.
Telomere function, cont’.
• Telomeres are also thought to be the "clock"
that regulates how many times an individual
cell can divide. Telomeric sequences
shorten each time the DNA replicates.
Telomerase
1. Telomerase binds to the telomer
and the internal RNA component
aligns with the existing telomer
repeats.
2. Telomerase synthesizes new
repeats using its own RNA
component as a template
3. Telomerase repositions itself on
the chromosome and the RNA
template hybridizes with the DNA
once more.
 and Primase
Structure of Telomeres
• Elongated strand of telomere repeats are
rich in guanine nucleotides
• 5’-TTTTGGGGTTTTGGGGTTTTGGGG-3’
• They have the capacity to hydrogen bond to
one another in the form of G-quartets.
• These create three-dimensional structures.
How Does Telomerase Work?
• Telomerase works by adding back
telomeric DNA to the ends of
chromosomes, thus compensating for the
loss of telomeres that normally occurs as
cells divide.
• Most normal cells do not have this enzyme
and thus they lose telomeres with each
division.
How Does Telomerase Work?
• In humans, telomerase is active in germ
cells, in vitro immortalized cells, the vast
majority of cancer cells and, possibly, in
some stem cells.
How Does Telomerase Work?
• Research also shows that the counter that
controls the wasting away of the telomere
can be "turned on" and "turned off". The
control button appears to be an enzyme
called telomerase which can rejuvenate the
telomere and allow the cell to divide
endlessly. Most cells of the body contain
telomerase but it is in the "off" position so
that the cell is mortal and eventually dies.
How Does Telomerase Work?
• Some cells are immortal because their
telomerase is switched on
• Examples of immortal cells: blood cells and
cancer cells
• Cancer cells do not age because they
produce telomerase, which keeps the
telomere intact.
Telomerase and Cancer
• There is experimental evidence from
hundreds of independent laboratories that
telomerase activity is present in almost all
human tumors but not in tissues adjacent to
the tumors.
Telomerase and Cancer
• Thus, clinical telomerase research is
currently focused on the development of
methods for the accurate diagnosis of
cancer and on novel anti-telomerase cancer
therapeutics
Experimentation
• Many experiments have shown that there is
a direct relationship between telomeres and
aging, and that telomerase has the ability to
prolong life and cell division.