DNA replication, repair and recombination Notes

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Part II Chapter 5 DNA Replication, Repair and Recombination
The maintenance of DNA sequence
• Mutation: permanent change in the DNA, it can destroy an organism if happens in vital location
• Mutation rate is one nucleotide change per 108 nucleotides per human generation. (70
nucleotides of each offspring)
• Mutation rate is extremely low.
• Germ cells: transmit genetic information from parent to offspring
• Somatic cells:transit genetic information form the body of the organism
• Change in somatic cells may lead to cancer
DNA replication mechanism:
• DNA polymerase catalyses the stepwise addition of a deoxynucleotide to the 3’ -OH end of the
polynucleotide chain
• The free nucleotide served as substrates for this enzyme were found to be deoxynucleotide
triphosphate
• The reaction is driven by a large favourable free-energy change, caused by the release of
pyrophosphate and its subsequent hydrolysis to two molecules of inorganic phosphate.
• The newly synthesised DNA strand therefore polymerised in the 5’-to-3’ direction only
• DNA polymerase performs the ﬁrst proofreading step just before a new nucleotide is added.
• The correct nucleotide has a higher afﬁnity for the moving polymerase than does the incorrect
nucleotide, because the correct pairing is more energetically favourable.
• After nucleotide binding, but before the nucleotide is covalently added to the growing chain, the
enzyme must undergo a conformational change in which its “grip” tightens around the active site.
It makes the nucleotide to double check the base-paired geometry
• The next error-correcting reaction, known as exonucleolytic proofreading, takes place
immediately after those rare instances in which an incorrect nucleotide is covalently added to the
growing chain.

• DNA molecules with a mismatched (improperly base-paired) nucleotide at the 3ʹ-OH end of the
primer strand are not effective as templates because the polymerase has difficulty extending
such a strand.
• DNA polymerase in this case will turn into editing mode, turning into 3’-to-5’ proofreading
exonuclease clipping off any unpaired or misfired nucleotides.
• DNA polymerase functions as a “self-correcting” enzyme that removes its own polymerisation
errors as it moves along the DNA
• If there were a DNA polymerase that added deoxyribonucleotide triphosphates in the 3ʹ-to-5ʹ
direction, would have to provide the activating triphosphate needed for the covalent linkage. In
this case, the mistakes in polymerisation could not be simply hydrolysed away, because the bare
5ʹ end of the chain thus created would immediately terminate DNA synthesis.
• Only DNA replication in the 5’-to-3’ direction allows efficient error correction
• DNA primases adds a RNA primer on the lagging strand for about 10 nucleotides long at the
intervals of 100-200 nucleotides
• Any enzyme that starts a new chain cannot be self-correct. So reason of using a RNA primer
instead of a DNA primer is to keep the accuracy of replication. DNA primer may make mistakes
without self-correcting mechanism. RNA primer will be efficiently removed ad replaced.

• DNA helicases were ﬁrst isolated as proteins that hydrolysed ATP when they are bound to single
strand of DNA. The hydrolysis of ATP can change the shape of a protein molecule in a
cyclical manner that allows the protein to perform mechanical work.
• Single-strand DNA-binding (SSB) proteins, also called helix-destabilizing proteins, bind
tightly and cooperatively to exposed single-strand DNA without covering the bases, which
therefore remain available as templates. These proteins are unable to open a long DNA helix
directly, but they aid helicases by stabilising the unwound, single-strand conformation.
• On their own, most DNA polymerase will synthesis only a short distance before falling off. This
property allows DNA polymerase to be recycled so quickly on the lagging strand
• A sliding clamp helps the DNA polymerase sticks ﬁrmly on the DNA while moving. The clamp is
a type of accessory protein (PCNA for eukayotes)
• The clamp has a large ring shape around the DNA double helix. The assembly of the clamp
requires ATP hydrolysis by a special protein, called clamp loader, which hydrolyses ATP as it
loads the clamp on to a primer-templates junction.
• In eukaryotes, Polδ completes each Okazaki fragment on the lagging strand and Polε extends
the leading strand.
• Summary: At the front of the replication fork, DNA helicase opens the DNA helix. Two DNA
polymerase molecules work at the fork, one on the leading strand and one on the lagging
strand. Whereas the DNA polymerase molecule on the leading strand can operate in a
continuous fashion, the DNA polymerase molecule on the lagging strand must restart at short

intervals, using a short RNA primer made by a DNA primase molecule. The close association of
all these protein components increases the efﬁciency of replication and is made possible by a
folding back of the lagging strand. This arrangement also facilitates the loading of the
polymerase clamp each time that an Okazaki fragment is synthesised: the clamp loader and
the lagging-strand DNA polymerase molecule are kept in place as a part of the protein machine
even when they detach from their DNA template.
• Strand-directed mismatch repair system detects the potential for distortion in the DNA helix
from the misﬁt between non complementary base pairs.
• In E.coli, methylation of all A residues in the sequence GATC is used to distinguish between the
old strand and the newly made strand.
• In eukaryotes, newly synthesized lagging-strand DNA transiently contains nicks (before they are
sealed by DNA ligase) and such nicks (also called single-strand breaks) provide the signal
that directs the mismatch proofreading system to the appropriate strand.
• Having a defective copy of mismatch repair genes in human can be very dangerous, it may lead
to hereditary nonpolyposis colon cancer (HNPCC) due to rapid accumulation of mutations.
• As replication fork moves along the DNA double helix, it creates a winding problem. For every 10
pairs of nucleotides being replication, one turn of the DNA helix must be completed. However,
turing the helix is energetically unfavourable, which will create overwound of DNA in front of
replication fork.
• DNA topoisomerase is used to solve this problem. It can be viewed as a reversible nuclease
that adds itself covalently to DNA backbone, breaking phosphdiester bond. The reaction is
reversible.

• Topoisomerase I produces a transient single-strand break, which allows two sections of DNA
rotates freely to each other.
• Topoisomerase II produces a transient double-strand break, it breaks one double helix
reversibly to create a gate, inducing a nearby DNA helix to pass through the gate and then closes
it.

The initiation and completion of DNA replication in chromosomes
• DNA replication begins at replication origin, where is rich of A-T base pairs due to weaker forces
between two strands (2 hydrogen bonds)
• Bacteria only has a single origin of DNA replication
• Replication origins attract initiator proteins that binds to double-stranded DNA and pull it apart
• In human, replication of average-sized chromosome will take 35 days if there is only a single
replication origin. In eukaryotes, there are multiple replication origin.
• DNA sequences that can serve as an origin of replication are found to contain: binding site for a
large, multisubunit initiator protein called ORC (origin recognition complex), a stretch of DNA
that is rich in As and Ts, at least one binding site for protein that facilitate ORC.
• In brief, during G1 phase, the replicative helicases are loaded onto DNA next to ORC to create a
prereplicative complex. Then, upon passage from G1 phase to S phase, specialized protein
kinases come into play to activate the helicases. The resulting opening of the double helix allows
the loading of the remaining replication proteins, including the DNA polymerases. The protein
kinases that trigger DNA replication simultaneously prevent assembly of new prereplicative
complexes until the next M phase resets the entire cycle
• This strategy provides a single window of opportunity for prereplicative complexes to form; thus
ensure all DNA is copied once only.
• Histone proteins are required to package DNA and they are usually made only in the S phage
during DNA replication.

• When a nucleosome is traversed by a replication fork, the histone octamer appears to be broken
into an H3-H4 tetramer and two H2A-H2B dimers
• The H3-H4 tetramer remains loosely associated with DNA and is distributed at random to one
or the other daughter duplex, but the H2A-H2B dimers are released completely from DNA.
• Freshly made H3-H4 tetramers are added to the newly synthesized DNA to fill into the “spaces,”
and H2A-H2B dimers—half of which are old and half new—are then added at random to com-
plete the nucleosomes
• As DNA polymerase δ discontinuously synthesizes the lagging strand, the length of each
Okazaki fragment is determined by the point at which DNA polymerase δ is blocked by a
newly formed nucleosome.
• This explains why the length of Okazaki fragments in eukaryotes (~200 nucleotides) is
approximately the same as the nucleosome repeat length.
• The orderly and rapid addition of new H3-H4 tetramers and H2A-H2B dimers behind a replication
fork requires histone chaperones (also called chromatin assembly factors).
• The histone chaperones, along with their cargoes (histone proteins), are directed to newly
replicated DNA through a specific interaction with the eukaryotic sliding clamp called PCNA
• These clamps are left behind moving replication forks and remain on the DNA long enough for
the histone chaperones to complete their tasks.
• When the replication fork reaches an end of a linear chromosome, the final RNA primer
synthesised on the lagging strand cannot be replaced by DNA because there is no 3’-OH end
available for repair polymerase, which means DNA will lost a part of its end during every DNA
replication
• In bacteria, circular DNA solves this problem. In eukaryotes, specialised nucleotide sequence at
the end of the chromosomes called telomeres can solved this problem. Telomeres contain many
tandem repeat, in human is GGGTTA
• Telomere DNA sequences are recognized by sequence-specific DNA-binding proteins that attract
an enzyme, called telomerase

• Telomerase recognises the tip of an existing telomere DNA repeat sequence and elongates it in
the 5ʹ-to-3ʹ direction, using an RNA template that is a component of the enzyme itself to
synthesise new copies of the repeat
• Telomeres must clearly be distinguished from these accidental breaks; otherwise the cell will
attempt to “repair” telomeres, causing chromosome fusions and other genetic abnormalities.
• A specialized nuclease chews back the 5ʹ end of a telomere leaving a protruding single-strand
end.
• This protruding end—in combination with the GGGTTA repeats in telomeres—attracts a group of
proteins that form a protective chromosome cap known as shelterin. In particular, shelterin
“hides” telomeres from the cell’s damage detectors that continually monitor DNA.
DNA Repair:
• Defeats in human repair genes can lead to some diseases due to high mutation rate such as
hereditary cool cancer, breast cancer and xeroderma pigmentosum (XP)
• DNA double helix can be damaged in many ways, including deprivation (loss of guanine),
deamination (cytosine to uracil), reactive metabolites, chemicals in the environment and
radiation.

Base excision repair: specific base change
• DNA glycosylase: recognise a specific type of altered base in DNA and catalyse its
hydrolytic removal, including: those that remove deaminated Cs, deaminated As, different types
of alkylated or oxidized bases, bases with opened rings, and bases in which a carbon–carbon
double bond has been accidentally converted to a carbon–carbon single bond
• AP endonuclease: cut the sugar backbone and add in nucleotides. Depurination can be
therefore directly repaired by AP endonuclease.
Nucleotide excision repair: distortion in double helix
• Excision nuclease finds out distortion in double helix, including covalent reaction between DNA
bases and large hydrocarbons and base dimer (T-T,C-T,C-C)
• DNA helicase cuts the section of distorted DNA out
• DNA polymerase rebuilds the double helix and DNA ligase ligates the helix
• Transcription-coupled excision repair: Nucleotide excision repair protein couples with
RNA polymerase which ensures the vital sections of genes are correct in gene
expression
• Translesion DNA polymerase is used in emergencies for replication highly-damaged
DNA sequences. However, they are not accurate as DNA polymerase due to lack of
exonucleolytic proofreading activity. They are only released in emergencies and make
“good guesses”.

Nonhomologous end joining:
• broken ends are simply brought together by DNA ligation
• quick and dirty
• deletion of DNA sequences occur at the site of ligation and will lead to loss of
nucleotides
• small amount of nucleotides loss is acceptable in mammalian somatic cells due to a
large genome
• mistakes can happen: broken chromosomes mistakenly covalently attach to another
Homologous recombination:
• accurately correct double stranded break
• homologous recombination often occurs just after DNA replication, when the two
daughter DNA molecules lie close together and one can serve as a template for repair
of the other.
• 5’ end of the damaged DNA is digested by specialised nuclease to produce
overhanging single-strand 3’ end
• Strand exchange: one of the single-strand 3ʹ ends from the damaged DNA molecule
worms its way into the template duplex and searches it for homologous sequences
through base-pairing.

• An accurate DNA polymerase extends the invading DNA strand using the information
form the undamaged strand.
• invading strand relates and reform the broken double helix.
• DNA synthesis continues using the strands from damaged DNA as templates
• DNA ligation to form complete double helix.
Strand Exchange:
• special protein does this job, in E Coli. is RecA and in all eukaryotes is Rad51
• RecA first binds cooperatively to the invading single strand, forming a protein–DNA
filament that forces the DNA into an unusual configuration: groups of three consecutive
nucleotides are held as though they were in a conventional DNA double helix but,
between adjacent triplets, the DNA backbone is untwisted and stretched out
• This unusual protein–DNA lament then binds to duplex DNA in a way that stretches the
duplex, destabilizing it and making it easy to pull the strands apart.
• The invading single strand then can sample the sequence of the duplex by conventional
base-pairing. This sampling occurs in triplet nucleotide blocks: if a triplet match is
found, the adjacent triplet is sampled, and so on.

• Homologous recombination can also repair replication fork.
• Replication fork may fall off due to nick or a gap in the parental DNA helix just ahead the
replication fork.
Regulate the use of homologous recombination:
• sometimes a broken human chromosome is repaired using the homolog from the other
parent instead of the sister chromatin
• maternal and paternal chromosomes differ in DNA sequence at many positions along
their lengths. Homologous recombination can convert the sequence of the repaired DNA
from the maternal to the paternal sequence or vice versa. This type of recombination is
known as loss of heterozygosity.
Homologous Recombination is crucial for meiosis:
• programmed double stranded break is preformed by a specialised protein (Spo11 in
budding yeast). Like a topoisomerase, Spo11 remains on the broken DNA sequence
• Specialised nuclease chews back at the 5’ end of the double helix, degraded the Spo11
and leaving a overhanging 3’ end
• Holiday junction (cross-strand exchange) is formed, two double-strand DNA helixes
are connected with speciﬁc protein, thereby stabilises the open symmetric isomers.

• Specialised proteins that bind to the holiday auctions can catalyse a reaction known as
branch migration, whereby DNA is spooled through the holiday auction by continually
breaking and reforming.
• Holiday auction therefore can move and expand the region of heteroduplex DNA from
initial site using the energy from ATP
• The outcome of the holiday junction can be non-crossover or crossover. 90% of
homologous recombination is non-crossover. But the crossover has signiﬁcant
meanings.
• We don’t know what decide crossover to happen. We know that crossover in one
position will inhibit crossover in the neighbouring regions. Crossover control ensures
the roughly even distribution of crossover points along the chromosomes.
• Roughly two crossovers occur per chromosome per mitosis
• In both crossover and non-crossover, recombination will leave a heteroduplex region
where a strand of paternal DNA is paired with a strand of maternal DNA. The regions can
last for thousands of nucleotides due to branch migration.

Gene conversion: If the two strands that make up a heteroduplex region do not have
identical nucleotide sequences, mismatched base pairs are formed, and these are often
repaired by the cell’s mismatch repair system. However, the mismatch repair system
cannot distinguish between the paternal and maternal strands and will randomly choose
the strand to be used as a template. As a consequence, one allele will be lost and the
other duplicated, resulting in net “conversion” of one allele to the other.
Transposition recombination:
• Mobile genetic element: a wide variety of specialised segments of DNA that can be
moved from one position in a genome into another
• Mobile elements that move by the way of transposition are called transposons, or
transposable elements
• In transposition, a specific enzyme, usually encoded by the transposon itself and
typically called a transposase, acts on specific DNA sequences at each end of the
transposon, causing it to insert into a new target DNA site.
• Most transposons move very rarely, in bacteria, transposons move once per 105 cell
division
• More frequent movement will probably destroy the cell genome.
• Transposons can be classified into DNA-only transposons, retroviral-like
retrotransposons, nonretroviral retrotransposons.
• DNA-only transposon: they exist only as DNA during their movement, predominate in
bacteria and they are largely responsible for the spreading of antibiotic resistance.
• DNA-only transposon can be relocated from the donor site to the target site by cut-and-
paste transposition. This reaction produces a short duplicated of the target DNA
sequence at the insertion site, which makes transposon inserted and ligated perfectly to
the insertion site. At both ends of transposon, short inverted repeat sequence are found
to indicated its identity.
• Double-stranded break cause by the loss of transposons can be repaired either by
homologous recombination or non-homologous end joining which will leaves a mutation
at the original transposon site.

• Certain viruses are considered mobile genetic elements because they use transposition
mechanism to integrate their genomes into that of their host cell.
• Retrovirus: exists as a single-stranded RNA genome packed into a protein shell along
with a virus-encoded reverse transcriptase enzyme
• The infection procedures of retrovirus involves turning single-stranded RNA into double
stranded DNA by reverse transcriptase, then virus-encoded transposase called
integrase inserts the viral DNA into the chromosome by a cut-and-paste transposition.
• Retroviral-like retrotransposons is relocated like retrovirus but lack of the protein coat.
• The ﬁrst step in their transposition is the transcription of the entire transposon, producing
an RNA copy of the element that is typically several thousand nucleotides long. This

transcript, which is translated as a messenger RNA by the host cell, encodes a reverse
transcriptase enzyme. This enzyme makes a double-strand DNA copy of the RNA
molecule via an RNA–DNA hybrid intermediate, precisely mirroring the early stages of
infection by a retrovirus. Then, the linear double-stranded DNA is inserted into the
chromosome by intergrase.
• Nonretroviral retrotransposon: distinct mechanism requires a complex of
endonuclease and reverse transcriptase
• A significant fraction o vertebrate chromosomes is made up of repeated DNA sequence.
In human, these repeats are mostly mutated version of nonretroviral retrotransposons
including LINE and SINE (long/short inter spread nuclear element)
• Some of transposition will lead to human diseases, for example, L1 insertion into gene-
coding blood-clotting protein factor VIII will cause haemophilia
Conservative site-specific recombination:
• Breaking and rejoining DNA sequence at two specific site.
• Depending on the position and orientation, it can be classified into DNA integration, DNA
excision and DNA inversion

• DNA virus can use this machismo to move their genome in and out the host cell easily.
• Conservative site-specific recombination can be also used in control of gene expression.
• Gene inversion can change the orientation of the promoter genes and therefore change
the gene expression. Due to reversibility, the gene on the both side and be switch on and
off easily.
Transposition Conservative site-specific recombination
requires only that the transposon have a
specialized sequence
requires specialized DNA sequences on
both the donor and recipient DNA
does not proceed through a covalently
joined protein–DNA intermediate
recombinases that catalyze conservative
site-specific recombination resemble
topoisomerases in the sense that they
form transient high-energy covalent
bonds with the DNA and use this energy to
complete the DNA rearrangements
leaves gaps in the DNA that must be
repaired by DNA polymerases.
No gaps

DNA replication, repair and recombination Notes

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