Replication, Maintenance, and

Rearrangements of Genomic
DNA
Introduction
Reproduction is a fundamental biological process.
Each time a cell divides, its entire genome must
be duplicated.
Complex enzymatic machinery is required to
copy the large DNA molecules. There are even
mechanisms to correct replication mistakes and
repair DNA damage.
But cell genomes are not static.
In order for species to evolve, mutations and
gene rearrangements are needed to maintain
genetic variation between individuals.
DNA Replication

DNA polymerase is the main enzyme in DNA

replication. It catalyzes the joining of
deoxyribonucleoside 5′- triphosphates (dNTPs).
It was first identified in 1956. It provided a
biochemical basis for the mode of DNA replication
that was initially proposed by Watson and Crick.
Both prokaryotic and eukaryotic cells contain several
different DNA polymerases that play distinct roles
in DNA replication and repair.
DNA Replication
All DNA polymerases share two fundamental properties:
• Synthesize DNA only in the 5′ to 3′ direction.
• Add new dNTPs only to a primer strand that is hydrogen-bonded to the template;
they can’t initiate DNA synthesis from free dNTPs.
DNA Replication
DNA replication was first analyzed by growing E. coli in the presence of
radioactive thymidine, which allowed visualization by autoradiography.
DNA molecules contained two replication forks, representing the regions
of active DNA synthesis.
DNA Replication
Since the two strands of DNA run in opposite (antiparallel) directions, only one strand
of DNA is synthesized in a continuous manner in the 5′ to 3′ direction (the leading
strand).
The other (lagging strand) is formed from short (1–3 kb), discontinuous pieces of
DNA that are synthesized backward.
These small pieces (Okazaki fragments) are joined by DNA ligase.
DNA Replication
Short fragments of RNA serve as primers for synthesis of Okazaki
fragments.
RNA synthesis can initiate de novo. An enzyme called primase synthesizes
short fragments of RNA that act as primers.
DNA Replication

The primers must then be removed and replaced

with DNA.
In prokaryotes, RNA primers are removed by
polymerase I, which acts as an exonuclease that
can hydrolyze DNA in either direction.
In eukaryotic cells, RNA primers are removed by
RNase H, which degrades the RNA strand of RNA-
DNA hybrids, and 5′ to 3′ exonucleases.
The resulting gaps are then filled by polymerase δ
and the fragments joined by DNA ligase.
Figure 6.5 Removal of RNA primers and joining of Okazaki fragments
DNA Replication
Different DNA polymerases have roles at the replication fork.
Eukaryotic cells have α, δ, and ε polymerases.
Polymerase α is found in a complex with primase.
DNA Replication
Other proteins at the replication fork:
• Proteins that bind to DNA polymerases and increase their activity and keep
them bound to the template DNA.
• Sliding-clamp proteins that load polymerase onto the primer and maintain its
stable association with the template, and clamp-loading proteins.
Figure 6.7 Polymerase accessory proteins (Part 2)
DNA Replication
Helicases catalyze the unwinding of parental DNA ahead of the replication
fork.
Single-stranded DNA-binding proteins then stabilize the unwound
template DNA so that it can be copied by the polymerase.
DNA Replication
As the DNA unwinds, the DNA ahead of the replication fork is forced to
rotate, which would cause circular DNA molecules to twist.
Topoisomerases catalyze reversible breaking and rejoining of DNA strands.
The transient breaks serve as swivels that allow the two strands to rotate
freely around each other.
DNA Replication
The enzymes involved in DNA replication act in a coordinated
manner to synthesize both leading and lagging strands of
DNA simultaneously.
DNA Replication

The accuracy of DNA replication is critical to cell reproduction.

Mutation rates indicate the frequency of errors during replication
is less than one incorrect base per 109 nucleotides.
This is much lower than would be predicted simply on the basis
of complementary base pairing.
DNA polymerase helps select the correct bases for insertion.
Binding of correctly matched dNTPs induces conformational
changes in DNA polymerase that lead to the incorporation of
the nucleotide.
This increases fidelity of replication about a thousand-fold.
DNA Replication
Proofreading:
DNA polymerases require primers and catalyze the growth of DNA strands only
in the 5′ to 3′ direction.
They also have exonuclease activity that can hydrolyze DNA in the 3′ to 5′
direction. When an incorrect base is incorporated, it is removed by the
exonuclease activity.
DNA Replication

Origins of replication are binding sites for proteins that initiate the
replication process.
In E. coli, an initiator protein binds to specific DNA sequences within the
origin.
The initiator protein begins to unwind the DNA and recruits other proteins
involved in DNA synthesis.
Helicase and single-stranded DNA-binding proteins then continue unwinding
and exposing the template DNA, and primase initiates synthesis.
Two replication forks are formed and move in opposite directions along the
circular chromosome.
Figure 6.12 Origin of replication in E. coli
DNA Replication
Multiple origins are needed to replicate the much larger genomes of
eukaryotic cells in a reasonable amount of time.
This was demonstrated by exposure of mammalian cells to radioactive
thymidine for different time intervals, followed by autoradiography to
detect newly synthesized DNA.
DNA Replication
Eukaryotic origins of replication were first studied in the yeast S. cerevisiae.
They were identified as sequences that can support replication of plasmids in
transformed cells.
Several such elements (autonomously replicating sequences, or ARSs)
have been isolated.
DNA Replication
Functional ARS elements include an 11-base-pair core sequence common to
many different ARSs.
The core sequence is the binding site of a protein complex (origin
recognition complex, or ORC) that is required for initiation of DNA
replication.
DNA Replication
The terminal sequences of linear DNA molecules
(telomeres) consist of tandem repeats of simple-
sequence DNA.
They are maintained by telomerase, which catalyzes
the synthesis of telomeres in the absence of a DNA
template.
Telomerase is a reverse transcriptase.
It carries its own template RNA, which is
complementary to the telomere repeat sequences.
Multiple copies of the telomeric repeat sequences can
be generated to maintain telomeres.
DNA Replication
The mechanism of telomerase action was determined in 1985 in studies of the
protozoan Tetrahymena.
The RNA template allows telomerase to extend the 3′ end by one repeat unit beyond
its original length.
The complementary strand can then be synthesized by the polymerase α-primase
complex.
DNA Replication

Removal of the RNA primer leaves an

overhanging 3′ end, which can form
loops at the ends of eukaryotic
chromosomes.

repeat fig 5.23 here

DNA Replication

Telomerase activity maintains telomeres at their normal

length.
Most somatic cells do not have enough telomerase to
maintain telomere length for an indefinite number of cell
divisions.
Telomeres gradually shorten as cells age, and this eventually
leads to cell death or senescence.
Several premature aging syndromes are characterized by an
abnormally high rate of telomere loss; some are caused by
mutations in telomerase.
Conversely, cancer cells express abnormally high levels of
telomerase, allowing them to continue dividing indefinitely.
DNA Repair
Damage to DNA can occur spontaneously or from exposure to
chemicals or radiation.
Damage can block replication or transcription, and lead to a
high frequency of mutations.
DNA Repair

Cells have evolved two categories of

DNA repair mechanisms:
• Direct reversal of the chemical
reaction responsible for DNA damage.
• Removal of damaged bases followed
by replacement with newly synthesized
DNA (excision repair).
DNA Repair
Only a few types of damage are repaired by direct reversal.
Exposure to UV light forms pyrimidine dimers, which distorts
the structure of the DNA and blocks transcription or replication
past the site of damage.
DNA Repair
Photoreactivation is one mechanism of repairing UV-induced pyrimidine
dimers.
Energy from visible light is used to break the cyclobutane ring structure,
reversing the dimerization reaction.
Many types of cells utilize photoreactivation but it is not universal; placental
mammals lack this mechanism of DNA repair.
DNA Repair
DNA damage can also result from alkylating agents—reactive
compounds that transfer methyl or ethyl groups to a DNA
base.
Methylation of the O6 position of guanine forms O6-
methylguanine, which forms complementary base pairs with
thymine instead of cytosine.
DNA Repair
This can be repaired by an enzyme (O6-methylguanine
methyltransferase) that transfers the methyl group from O6-
methylguanine to a cysteine residue in its active site.
DNA Repair

Excision repair can be used for a wide

variety of DNA damage.
There are three types:
• Base-excision
• Nucleotide-excision
• Mismatch repair
DNA Repair

In base-excision repair, single damaged bases are recognized and

removed.
Example: uracil can arise in DNA when dUTP is incorporated in place of
thymine, or uracil can be formed in DNA by the deamination of cytosine.
Excision of uracil in DNA is catalyzed by DNA glycosylase, which cleaves
the bond between the uracil and the deoxyribose of the DNA.
DNA glycosylases also recognize and remove other abnormal bases.
An apyrimidinic or apurinic site (AP site) is a sugar with no base attached;
formed by excision of a base.
Purine bases can also be lost spontaneously.
These sites are repaired by AP endonuclease. The deoxyribose is
removed, and the resulting gap is filled by DNA polymerase and ligase.
Figure 6.21 Base-excision repair
DNA Repair
Other excision repair systems recognize a wide variety of
damaged bases, including bulky groups added to DNA by
carcinogens.
DNA Repair
Nucleotide-excision repair removes the damaged bases as part of an
oligonucleotide.
An excinuclease is an enzyme complex that can directly excise an oligonucleotide.
Helicase is required to unwind the DNA for excision; the resulting gap is filled by DNA
polymerase and sealed by ligase.
DNA Repair
Human DNA repair genes have been identified in studies of
inherited diseases.
Xeroderma pigmentosum (XP) causes extreme sensitivity to
UV radiation and multiple skin cancers; it results from lack of
nucleotide-excision repair.
XP cells are used as an experimental system to identify DNA
repair genes.
Molecular cloning has identified different repair genes that are
mutated in individuals with XP and other diseases.
The encoded proteins are closely related to proteins encoded
by yeast RAD genes, indicating that nucleotide-excision
repair is highly conserved.
DNA Repair
The proteins are used in vitro systems to study their roles in
the repair process.
Repair genes mutated in XP are designated XPA through
XPG.
Disrupted base pairing is recognized by XPC, followed by
the cooperative binding of XPA, RPA, and a transcription
factor called TFIIH.
Two subunits of TFIIH are the XPB and XPD proteins,
which act as helicases to unwind about 25 base pairs
around the site of damage.
XPG protein is then recruited to the complex followed by
XPF/ERCC1. These are endonucleases that cleave DNA
on the 5′ and 3′ sides of the damaged region.
Figure 6.23 Nucleotide-excision repair in mammalian cells (Part 1)
DNA Repair

Transcription-coupled repair is nucleotide-

excision repair during transcription.
When RNA polymerase is stalled by DNA
damage in mammalian cells, it is recognized
by two proteins (CSA and CSB) that then
recruit other proteins to repair the damage.
In patients with Cockayne’s syndrome, genes
for CSA and CSB are mutated, and they are
defective in transcription-coupled repair.
Figure 6.24 Transcription-coupled repair in mammalian cells (Part 1)