DNA Synthesis
• Synthesis of polynucleotide chain by DNA Polymerase requires:
o ATP
o Magnesium ions Mg2+
o Nucleoside triphosphates
• Polymerases always work in the 5’ to 3’ direction (fill in)
DNA Damage and Repair
Chemical Damage
1. Pyrimidine dimers
• Cause: UV light
• 2 adjacent pyrimidines joined by a cyclobutene ring (phosphodiester bond)
• 2 pyrimidines are close together and when UV comes along, they can form a chemical reaction
• Lesion is formed on DNA because the 2 bases are chemically coupled together
• Repair:
o Direct Reversal (For Prokaryotes only)
▪ Repair of pyrimidine dimers
▪ Using photoreactivation enzymes (Humans do not have this [1])
▪ Uses visible light to break cyclobutene ring
▪ Only in yeast, plant and animals, not humans
▪ Humans do repair thymidine dimers but in a different way
o Nucleotide Excision Repair (Type of Excision Repair) – in Eukaryotes
1. Damaged DNA (Thymidine dimer) is recognized
2. Bubble forms
3. Damaged DNA is cleaved on both sides of the thymine dimer by 3’ and 5’ endonucleases.
Endonucleases cut into the DNA whereas an exonuclease cuts from the end of the DNA.
4. So anyway now you end up with nicks in the DNA
5. Unwinding by a helicase results in excision of an oligonucleotide containing the damaged bases (aka
the TT dimer). So now you’re left with a gap on the DNA with lots of single-stranded bases acting as
a template strand
6. The resulting gap is then filled by DNA polymerase and sealed by ligase. DNA pol, in a 5’ to 3’
direction, carries out a fill in reaction of DNA synthesis until it reaches the other side of the DNA.
Ligase repairs the phosphodiester backbone.
▪ Larger than Base Excision Repair
▪ Removes entire oligonucleotide (a nucleotide stretch around the damage), leaving a gap in 1 strand
▪ You have to remove an entire single strand oligonucleotide stretch and replace it using Fill-in of DNA
synthesis
▪ In theory, every reaction is reversible. By chance, it could get ligated back but then the machinery
would detect a lesion and re-repair it. But it’s very unlikely that this be ligated back in because the
endonuclease removes this very quickly and there is a higher entropy (more energetically favorable)
to remove thymidine dimers.
▪ Present in E.coli
- Catalysed by 3 gene products – uvr (ultraviolet radiation) A, B, C
- Mutations of these genes leads to high sensitivity to UV, much more prone to being killed by
UV light
- UvrA recognises damaged DNA, UvrB and UvrC are the endonucleases that form a complex
with the uvr A and cleave at 3’ and 5’ sides, leading to a 12-13 bases oligonucleotide being
cleavable and excisable from the cell
▪ Present in Humans
, - More complicated
- Catalysed by RAD gene products in yeast (radiation sensitivity)
- Disease associated: Xeroderma Pigmentosum
• rare genetic disorder
• affects 1:250,000 people
• extreme sensitivity to UV light, skin cancers if left untreated, scarring on skin
• deficient in ability to repair DNA by nucleotide-excision
• 7 different repair genes involved – highly conserved across most humans bc they
fulfil such an essential task in DNA repair
2. Alkylation
• Spontaneous damage
• Addition of methyl/ethyl groups to various positions on the DNA
• Example: Guanine to O6-methylguanine, O6-methylguanine base pairs with thymine instead of cytosine
• Changes hydrogen bonding potential
• Repair:
o O6-methylguanine methyltransferase reverses the process
▪ Widespread in prokaryotes and eukaryotes
▪ Active site of O6-methylguanine methyltransferase contains this reactive amino acid cysteine which
has an SH group that reacts with the methyl group and rips it off the guanine in order to restore the
original carbonyl group on the guanine, leaving the methyl group covalently attached to the
methylcysteine
▪ One step process
▪ Enzyme is inactive in this form and has to be regenerated with chemical reactions that remove the
methyl groups so that the enzyme can be recycled and react again
3. Bulky carcinogens
• Cause: burnt meat, or activated endogenously by cellular enzymes like cytochrome P450
• Ex: benzo-(a)pyrene
• Addition of large bulky chemical groups to DNA
• All functional groups can be carcinogens
o They don’t even have to be covalently interacting (no reactive functional group).
o Intercalating agents sit in the DNA bases and don’t covalently react but they mess up the DNA formation
• Forms bulky lesions on DNA
• Activated endogenously by cellular factors like reactions with cytochrome P450
o Cells use cytochrome P450 as molecular dustbins to degrade and remove toxins but sometimes, they make
reactive intermediates instead.
o Reactive intermediates can covalently react with DNA
Spontaneous Damage
1. Deamination
• Removal of amine group
• Happens to adenine, cytosine and guanine
• Changes hydrogen bonding potential
• A changes to G, C to U and G to ..
o Adenine contains an amine group (Hydrogen donor) on the major groove and when deaminated, it
forms a C=O bond (Hydrogen acceptor), forming the base Hypoxanthine
o Hypoxanthine looks like Guanine and so base pairs with Cytosine
• Causes bulge/lesion
• Repair
, o Base Excision Repair (Both in Bacteria and Humans) using the example of cytosine being deaminated to
uracil
1. Uracil formed by deamination of cytosine, forming G:U mismatch
2. The mismatch caused a bulge or a ‘lesion’ within the double stranded DNA
3. The lesion and/or bond between uracil and deoxyribose is recognized by DNA glycosylase
4. DNA glycosylase removes the damaging base pair so you have a specific DNA Glycosylase for every
base.
5. For uracil, Uracil DNA glycosylase cleaves the bond and leaves a sugar with no base attached in the
DNA (an AP site). The phosphodiester backbone is intact and just missing a base (AP site).
6. The AP site is recognized by AP endonuclease which cleaves the DNA chain and makes a nick.
7. Remaining deoxyribose sugar (backbone) is removed by deoxyribose-phosphodiesterase which
hydrolyses the phosphate sugar contact on that base.
8. What you’re left with then is a single gap in the DNA which would be the perfect recipient for a new
nucleoside triphosphate component such as dATP or in this case bc it’s a guanine, a dCTP.
9. This gap is then filled in with a DNA polymerase which puts cytosine opposite the G so it can
correctly be repaired.
10. But then there is a gap left on one side of the sugar phosphate backbone and that side has to be
joined together with a DNA Ligase.
2. Depurination
• Removal of purine group
• Results in cleavage of bond between purine base and deoxyribose, leaving AP (apurinic) site in DNA
• Changes hydrogen bonding potential and base pairing potential so wrong watson-crick base pairing
DNA Polymerase Error Damage – Mismatch Repair
• In E.coli:
o How to distinguish between parental and daughter strand?
▪ Daughter strand is hemi-methylated, parent strand is fully
▪ E.coli contains Dam methylase, which the E.coli uses naturally to protect its DNA from its own
restriction enzymes that it uses as a defense mechanism to kill foreign invading DNA
▪ So when the phage virus infects the cell, its restriction enzymes will cut the foreign DNA bc it’s not
methylated but not its own DNA bc it’s methylated
▪ Newly synthesized daughter strand has not had sufficient time to be recognized by Dam methylase
and methylated so the new strands do not contain methylation sites temporarily. They will be hemi-
methylated (half methylated)
o In vitro assays are used to assess the repair of different substrates
o Happens post replication by DNA pol
o Involves MutH, MutS, MutL, ATPase activity
1. GT mismatch occurs due to DNA pol’s mistake
2. Bulge formed
3. MutS recognizes bulge and recruits MutL
4. MutL binds to MutS
5. MutL has ATPase activity for forming DNA loops and allow it to translocate along the DNA looking for hemi-
methylated Dam sites
6. When it finds a hemi-methylated site, MutH binds to MutL, activating MutH
7. MutH (endonuclease) recognizes the non-methylated strand and nicks that site (can be upstream or
downstream of mismatch)
8. Depending on whether the nick occurs on one side or the other, bc of the orientation of the 5’ to 3’ ends of
the open DNA strands, you require diff exonucleases to come in and chew away the bad DNA. Different
exonucleases required depending on polarity
9. If it occurs on a cis (I think) site, you recruit either an exonuclease VII or a RecJ enzyme.
, 10. The exonuclease chews back the DNA in 5’ to 3’ direction in order to remove the mismatch until the lesion is
removed.
11. Once it’s removed, DNA pol III come in and fill in the gap, replacing the mismatch with the correct base and
then the final phosphodiester break is reconnected using Ligase
12. Similarly, on the other side, you need a diff exonuclease to chew the DNA from a diff side, this time u have a
3’ to 5’ exonuclease (Exo I).
13. Once you go past the lesion, then the problem has been removed. Again DNA pol fill in in the 5’ to 3’
direction and the final break is fixed w ligase.
• In Mammals:
o Also occurs after DNA synthesis but mammals do not have the same methylation mechanism
o Similar to E. coli, except that the newly replicated strand is distinguished from the parental strand because it
contains strand breaks and not Dam methylated
o Since eukaryotic DNA contains many replicons which means that eukaryotic chromosomes are synthesized
on both strands multiple times simultaneously during DNA synthesis. So you end up with Okazaki fragments
on both new strands
o Enzymes involved:
▪ MutS, MutL, helicase, exonuclease, DNA polymerase, ligase
1. Mismatch base on the new strand which is recognized
2. MutS and MutL comes along and recognizes that mismatch, recruits helicase and exonuclease to chew
away the new strands until the lesion is removed.
3. DNA pol and ligase fill in and join the gap.
o Disease: Non-polyposis colorectal cancer
▪ Mutations in hMsh2 and hMIh1 genes
Double-stranded Break Repair
• Involves 2 nicks, end up with ds break
• Occur due to DNA replication, X-ray damage or nucleases
• DNA lesions lead to the stalling of DNA polymerases during recombination
• DNA nicks lead to an end of DNA pol copying DNA
• Both events can lead to this occurrence of dsDNA break ends
• Repair:
1. Non-Homologous End Joining (opposite to Homologous Recombination) NHEJ
o No homologous template
o Mostly in eukaryotes
o Prone to error and often introduces insertions and deletions
o Resection of ends then ligation
o Happens a lot in eukaryotes but not only found in eukaryotes
o Associated to Vdj recombination of immune system
▪ Vdj recombination increases the error rates of recombination and mutation and variability of
antibody repertoires in the immune system
▪ Generates diversity for immune system bc error-prone
o Used in CRISPR/Cas9 as a way to knock out genes
o NHEJ outcomes are often related to micro-homologies in DNA ends [1]
2. Homologous Recombination
o Not prone to error
o Requires homologous DNA for template
o Contributes to much of the variation in offspring = Evolution
▪ In meiosis, you have targeted HR for gene shuffling to scramble maternal and paternal
chromosomes leading to non-parental combinations (so you don’t look exactly the same as
1 of your parents)
