CHAPTER 13
DNA Replication and Repair
Introduction
• Reproduction is a property of all
organisms.
• DNA duplicates by a process called DNA
replication.
• The DNA replication machinery is also
used for DNA repair.
13.1 DNA Replication (1)
• DNA replication takes
place by separation of
the strands of the
double helix, and
synthesis of two
daughter strands
complementary to the
two parental
templates.
DNA Replication (2)
• Semiconservative Replication
– DNA replication is called semiconservative
because half of the parent structure is
retained in each of the daughter duplexes.
– This model of DNA replication took over the
other tow models previously considered:
conservative and dispersive.
Three alternate schemes of replication
Three alternate schemes of replication
Three alternate schemes of replication
DNA Replication (3)
• The Messelson and Stahl experiments
supported the semiconservative model of
replication in bacterial cells.
• Semiconservative replication was later
demonstrated in eukaryotic cells.
Experimental demonstration of semiconservative
DNA replication in bacteria
Experimental demonstration of semiconservative
DNA replication in eukaryotes
DNA Replication (4)
• Replication in Bacterial Cells
– Temperature-sensitive (ts) mutants were
used to identify the genes of replication.
– Replication can be studied using in vitro
systems reconstituted from purified cellular
compounds.
DNA Replication (5)
• Replication Forks and Bidirectional
Replication
– Replication starts at the origin site, where a
number of proteins bind to initiate replication.
– Replication proceeds bidirectionally.
– Replication forks are points where a pair of
replicating segments come together and join
the nonreplicated segments.
Model of a bacterial chromosome
undergoing bidirectional replication
DNA Replication (6)
• Unwinding the Duplex and Separating the
Strands
– Tension is built up as DNA begins the
unwinding process, and the DNA becmes
positively supercoiled.
– DNA gyrase (topoisomerase II) relieves the
tension by changing the DNA into negatively
supercoiled DNA.
The unwinding problem
DNA Replication (7)
• The Properties of DNA Polymerases
– DNA polymerase is responsible for
synthesizing new DNA strands from a DNA
template.
– DNA polymerase requires a primer which
provides the 3’ hydroxyl terminus on which to
add new nucleotides.
– Polymerization occurs in the 5’-to-3’ direction.
– None of the three DNA polymerases in
bacteria can initiate DNA chains.
Templates and nontemplates for DNA
polymerase activity
DNA Replication (8)
• Semidiscontinuous Replication
– Both daughter strands are synthesized
simultaneously.
– The leading strand (in the direction of the
replication fork movement) is synthesized
continuously.
– The lagging strand (in the opposite direction
of the replication fork movement) is
synthesized discontinuously.
The incorporation of nucleotides onto the 3’
end of a growing strand by DNA polymerase
The two strands of a double helix are synthesized
by a different sequence of events
DNA Replication (9)
• The lagging strand is
constructed of small
Okazaki fragments,
which are joined by
DNA ligase.
DNA Replication (10)
• Primase is an RNA
polymerase that
assembles short
RNA primers.
• These primers are
later removed and
the gaps are sealed.
DNA Replication (11)
• The Machinery Operating at the
Replication Fork
– Helicase and single-stranded DNA-binding
(SSB) proteins unwind the parental duplex
and separate the two strands.
– Primase and helicase form a “primosome”,
which processively moves along the laggingstrand template.
– A single replisome synthesizes both leading
and lagging strands.
The role of DNA helicase, SSB proteins, and
primase at the replication fork
The role of DNA helicase, SSB proteins, and
primase at the replication fork
DNA Replication (12)
• The Structure and Functions of DNA
Polymerases
– DNA polymerase III is the primary replication
enzyme.
• DNA polymerase III holoenzyme contains various
subunits having different functions in the
replication process.
• By forming a β clamp, one of the components
maintains an association between the polymerase
and the DNA template.
Replication in E. coli by DNA polymerases
working together as part of a complex
DNA Replication (13)
• As long as it is attached to a β “sliding
clamp”, DNA polymerase can move
processively from one nucleotide to the
next.
• The assembly of the β clamp around the
DNA requires a clamp loader, which is part
of the DNA polymerase III holoenzyme.
Schematic representation of
DNA polymerase III
The β sliding clamp and clamp loader
DNA Replication (14)
• Exonuclease Activities of DNA
Polymerases
– DNA polymerase I is involved in DNA repair
and also removes RNA primers and replaces
them with DNA.
– Exonucleases degrade nucleic acids by
removing 5’ or 3’ terminal nucleotides.
The exonuclease activities of
DNA polymerase I
DNA Replication (15)
• Ensuring High Fidelity during DNA
Replication
– The error rate of incorporation of an incorrect
nucleotide during DNA replication is the
spontaneous mutation rate.
– Incorporation of a particular nucleotide onto
the end of growing strand depends upon the
geometry of the base pair.
Geometry of proper and
mismatched base pairs
DNA Replication (16)
• Ensuring high fidelity (continued)
– During proofreading, mismatched bases are
excised.
– Careful selection of the nucleotide,
proofreading, and mismatch repair account for
low error rates in replication (about 10–9).
– Replication is rapid (~103 nucleotides/sec).
Activation of the 3’  5’ exonuclease activity
of DNA polymerase I
DNA Replication (17)
• Replication in Eukaryotic Cells
– Replication is eukaryotes is not as well
understood as replication in bacteria. Some
advances include:
• Using mutant yeast cells unable to produce
specific gene products for replication.
• Development of in vitro systems where replication
can occur in cellular extracts or mixtures of purified
proteins.
DNA Replication (18)
• Initiation of Replication in Eukaryotic Cells
– Eukaryotes replicate their genome in small portions
(replicons).
– Initiation of DNA synthesis in a replicon is regulated.
DNA Replication (19)
• Origins of replication identified in yeast
cells are called autonomous replicating
sequences (ARS).
• A multiprotein origin recognition complex
(ORC) is assembled at the ARS.
• Replication in mammalian cells has been
more difficult to study.
Replication of a
yeast replicon
DNA Replication (20)
• Restricting Replication to Once Per Cell
Cycle
– Replication origins pass through different
states so that they only replicate their DNA
once during a cell cycle.
• Origin of replication bound by an ORC.
• Proteins called “licensing factors” bind to the origin.
• Activation factors bind to the chromosomes and
induce “licensed” origins to begin replication.
DNA Replication (21)
• The Eukaryotic Replication Fork
– Replication activities are similar in eukaryotes
and prokaryotes.
– There are several DNA polymerases in
eukaryotes.
– Eukaryotic DNA polymerases elongate in the
5’-to-3’ direction and require a primer; some
have 3’-to-5’ exonuclease activity.
Some Proteins Required for Eukaryotic
DNA Replication
Schematic view of the major components of
the eukaryotic replication fork
DNA Replication (22)
• Replication and Nuclear Structure
– The replication machinery is stationary in the
nuclear matrix.
– Replication forks are located within sites
called replication foci.
– The clustering of replication forks may provide
a mechanism for coordinating replication of
adjacent replicons on individual
chromosomes.
The involvement of the nuclear matrix
in DNA replication
Demonstration that replication activities are
confined to distinct sites
DNA Replication (23)
• Chromatin Structure and Replication
– The assembly of DNA into nucleosomes is a
rapid event.
– Histones remain intact during replication and
old and new histones are distributed randomly
between the two daughter duplexes.
– The assembly of nucleosomes is facilitated by
a network of accessory proteins.
The distribution of histone core complexes
to daughter cells following replication
13.2 DNA Repair (1)
• DNA repair is essential for cell survival.
– DNA is the cell molecule most susceptible to
environmental damage.
– Ionizing radiation, common chemicals, UV
radiation and thermal energy create
spontaneous alteration (lesions) in DNA.
– Cells have a number of mechanisms to repair
genetic damage.
A pyrimidine dimer that has formed within a
DNA duplex following UV irradiation
DNA Repair (2)
• Nucleotide Excision Repair
– Nucleotide excision repair (NER) removes
bulky lesions, such as pyrimidine dimers and
chemically altered nucleotides.
– It consists of two pathways:
• A transcription-coupled pathway which is the
preferential pathway and selectively repairs genes
of greatest importance to the cell.
• A global genomic pathway which is less efficient
and corrects DNA strands in the remainder of the
genome.
Nucleotide excision
repair
DNA Repair (3)
• Nucleotide excision repair (continued)
– TFIIH is a key component of the repair
machinery and is also involved in the initiation
for transcription. It links transcription and DNA
repair.
– A pair of endonucleases cut on both sides of
the lesion, and the damaged strand is
removed by helicase.
– The gap is filled by a DNA polymerase and
sealed by DNA ligase.
DNA Repair (4)
• Base Excision Repair
– Base excision repair (BER) removes altered
nucleotides that produce distortions of the
double helix.
– DNA glycosylase recognizes the alteration
and cleaves the base form the sugar.
– DNA glycosylases are specific for a particular
type of altered base.
Base excision repair
Detecting damaged bases during BER
DNA Repair (5)
• Base excision repair (continued)
– DNA glycosylase removes the altered bases.
– Once the altered base is removed, an
endonuclease cleaves the DNA backbone
and a polymerase fills the gap by inserting a
nucleotide complementary to the undamaged
strand.
– The strand is sealed by DNA ligase.
DNA Repair (6)
• Mismatch repair (MMR) is the correction
of mistakes that escape the DNA
polymerase proofreading activity.
– Repair enzymes recognize distortions caused
by mismatched bases.
– In bacteria, the parental strands are
recognized from daughter strands by the
presence of methylated bases.
– Several MMR pathways have been identified
in eukaryotes.
DNA Repair (7)
• Double-Strand Breakage Repair
– Ionizing radiation (X-rays, gamma rays) along
with some chemicals cause double-strand
breaks (DSBs).
– DSBs can be repaired by a pathway in
mammalian cells called nonhomologous end
joining (NHEJ) in which proteins bind to the
broken ends and catalyze reaction to rejoin
the broken ends.
Repairing DSBs by NHEJ
DNA Repair (8)
• Double-strand breakage repair (continued)
– Cells that lack one of the proteins required for
NHEJ are very sensitive to ionizing radiation.
– Another DSB repair pathway is homologous
recombination, and requires a homologous
chromosome to serve as a template for repair
of the broken strand.
– Defects in both repair pathways have been
linked to increased cancer susceptibility.
The Human Perspective: The Consequences of
DNA Repair Deficiencies (1)
• Xeroderma pigmentosum (XP) patients
cannot repair sun-damaged DNA via NER.
• Some help for XP patients may become
available in the form of skin creams that
contain DNA repair enzymes.
• Cockayne syndrome (CS) patients re
deficient in the pathway by which DNA that
is transcriptionally active is repaired.
Xeroderma pigmentosum
The Human Perspective: The Consequences of
DNA Repair Deficiencies (2)
• Skin cells with optimal levels of repair
enzymes are subject to lesions that fail to
be excised and repaired.
• Skin cancer is not the only disease
promoted by deficiency or overworked
DNA repair systems.
• Some colon cancer cases are due to
mutations in mismatch repair genes.
13.3 Between Replication and
Repair
• Sometimes a DNA lesion is not repaired before
DNA is replicated by recruiting a special
polymerase that bypasses lesions.
• When polymerase η replicates stretches of
damaged DNA, DNA synthesis continues.
• DNA polymerase η engages in translesion
synthesis (TLS).
• TLS polymerases accommodate altered
nucleotides that would not fit within a classic
polymerase and have no proofreading activity.