DNA damage

Mutations in germ cells

Propagated through subsequent generations

Somatic cell mutations

Not passed to offspring but can lead to disease states (like cancer)

Point mutations

are changes in a single nucleotide base pair in the DNA sequence, which can result in silent, missense, or nonsense mutations.

Point mutation- transition

Purine to purine or pyrimidine to pyrimidine

Point mutation A—>G or C—> T are both examples of what type of mutation and why

Transition because they involve a change from a purine to a purine or vice versa

Point mutation- transversion

Involves a change from a purine to a pyrimidine or vice versa.

Point mutation from C—G or A—T would be what type of mutation

Transversion, as it involves a change between a purine to a pyrimidine or vice versa

Is a transition or transversion mutation more likely to occur

Transition because they are less likely to result in AA substitutions

Stop codons

UAA, UAG, UGA

Insertion or deletion of 1-2 nucleotide

Frameshift mutation- drastic change in AAs

Insertion/deletion of 3 nucleotides

Still could impair protein function, but non frameshift

Which codon position is most likely to cause a silent mutation

The third codon position, due to the redundancy of the genetic code.

What are the biological causes of mutations

Replication errors
Slippage

Slippage during replication

DNA polymerase encounters a secondary structure in the DNA, causing it to temporarily dissociate from the template strand, leading to the addition or deletion of nucleotides, which can result in frameshifts or other mutation

Backward slippage during replication

occurs when the DNA polymerase synthesizes new DNA and mistakenly reverts to a previous position on the template strand, resulting in the insertion of extra nucleotides.

Forward slippage during replication

occurs when DNA polymerase adds nucleotides to the growing strand while the template strand temporarily loops out, causing deletions in the newly synthesized DNA.

What is Huntington’s disease an example for

DNA slippage adds CAG (Glutamine) repeats into the gene causing Huntington’s disease

Why are the extra Gln residues so bad in Huntington’s disease

cause abnormal protein aggregates, which cause plaque on neurons causing neurodegeneration.

Inheritance of Huntington’s

is autosomal dominant, meaning that only one copy of the mutated gene from an affected parent can lead to the disease in offspring.

Alkylation of DNA

is a form of DNA damage where methyl/ethyl groups are added to keto residues

Consequences of DNA alkylation

Formation of methoxy will cause base-pairing errors and potentially lead to mutations or cell death.

Medical use of alkylating agents

Chemotherapeutic drug to target replicating cells

Weapon use of alkylating agents

Mustard gas has mutagenic effects

Depurination

is the loss of purine bases (adenine or guanine) from DNA

How does depurination occur

Base is removed by nucleophilic attack

What conditions favor depurination

Sporadic but favored by acidic conditions

Is depurination or depyrimidination more common

Depurination

Deamination

Removal of an amino group

Deamination of DNA can occur in which bases

Cytosine, adenine, and guanine (CAG)

Which base is most commonly affected by deamination

Cytosine —> uracil.

What happens if adenine or guanine are deaminated

Unusual bases like hypoxanthine and xanthine are formed, which cannot perform normal base pair activities

What conditions favor deamination

Sporadic or facilitated by deaminase enzyme

Oxidative DNA stress in induced by

exposure to radiation
 cigarette smoke or other tobacco products
 alcohol consumption
 diets high in fat, sugar, and processed foods

Most frequent oxidative DNA damage

formation of 8-Oxo-2'-deoxyguanosine (8-oxo-dG) after hydroxyl radicals attack guanine

(8-oxo-dG) binds to

adenine during DNA replication, leading to G:C to T:A transversions.

Thymine dimers

are formed when DNA is exposed to ultraviolet light, leading to covalent bonding between adjacent thymine bases, disrupting normal base pairing and causing mutations.

Sun burn is caused by

thymine dimers which induce apoptosis and production of protective melanin

Chromosome translocation

Chromosome segments are exchanged between two different chromosomes leading to derivatives

Base analog

is a chemical compound that resembles a normal DNA base and can be incorporated into DNA during replication, potentially causing mutations.

5-bromo uracil

base analog that can base pair with purines (adenine or guanine) and cause genetic defects

5-bromo uracil is used as a

chemotherapeutic agent to treat cancer by interfering with DNA replication.

Intercalating agent

have flat planar structures and can insert between base
stacks in the dsDNA helix causing distortions which interfere with normal replication

Examples of intercalating agents

include ethidium bromide and proflavine

Direct damage from ionizing radiation

Double and single stranded breaks in DNA

Indirect damage from ionizing radiation

occurs when ionizing radiation generates reactive oxygen species that damage DNA, leading to oxidative stress and potential mutations.

g1 checkpoint

prevents the replication of damaged genomic
material by blocking entry into S phase

S checkpoint

responds to the presence of DNA damage and
to aberrant replication forks by stopping or slowing DNA
synthesis

G2 checkpoint

impedes cells with damaged or
entangled/catenated DNA from undergoing mitosis

Spindle checkpoint

only allows mitotic exit if the chromosomes are
properly attached to the mitotic spindle

Progression through checkpoints are mediated by

various cyclin-dependent kinases (CDKs) and cyclins that regulate the cell cycle.

p53

MAJOR ‘guardian’ of our genome integrity; tumor suppressor

Which proteins are activated in response to DNA damage

ATM and ATR

What do ATM and ATR do

upregulate checkpoint proteins Chk2 and
Chk1, respectively

Function of Chk1 and 2

are to regulate the cell cycle and ensure DNA repair processes are activated in response to DNA damage.

How are thymine dimers repaired

Photorepair by DNA photolyase

What happens during Photorepair by DNA photolyase

light activated enzyme that can
reverse the covalent bond between thymine residues
with energy transfer via FADH-/FADH+ (flavin)

O6-methylguanine

is a DNA lesion resulting from the alkylation of guanine, leading to mispairing and potential mutations.

How is O6-methylguanine repaired

Methylguanine methyltransferase
(MGMT) has a specific cysteine residue
that can remove the methyl group and
convert it back to a keto group

Why is MGMT considered a suicide enzyme

because it irreversibly inactivates itself after removing the methyl group from O6-methylguanine, preventing further catalytic activity.

DNA Nick Repair

single stranded breaks from ionizing radiation are repaired by ligase

Base excision repair

is a cellular mechanism that repairs damaged DNA by removing and replacing incorrect or damaged bases.

Base excision repair prokaryotes

Glycosylase cuts out base; leaves backbone intact
 AP endonuclease introduces nick at that site
 DNA Pol I replaces nucleotide (plus surrounding
nucleotides)
 ligase seals the remaining nick

Base excision repair eukaryotes

DNA Polymerase can displace former strand
and flap endonuclease removes flap

Or Pol β can replace the damaged nucleotide
and add the correct one

E coli nucleotide excision repair system (NERS) is used for

Bulkier damage (not just a single base)

E. coli NERS step 1

UvrA and uvrB bind to lesion

E. coli NERS step 2

UvrC endonuclease generates a nick downstream of lesion

E. coli NERS step 3

DNA pol I fills in gap and ligase repairs nick

Eukaryotic nucleotide excision repair system (NERS) step 1

XPC recognizes damage/lesions and recruits two other
proteins (XPB and XPD)

Eukaryotic NERS step 2

XPB and XPD recruit the endonucleases XPF and XPG,
which generate nicks surrounding the damaged area

Eukaryotic NERS step 53

DNA polymerase fills in the gap and ligase seals the final nick.

E coli mismatch repair

is a process that corrects errors that occur during DNA replication, specifically targeting base pair mismatches and insertion-deletion loops.

E. coli mismatch repair step 1

MutS scans DNA, recognizes mismatches from the distortion caused in DNA backbone

E. coli mismatch repair step 2

The complex of MutS and the mismatch carrying DNA recruits
a second protein, MutL

E. coli mismatch repair step 3

MutL activates MutH, an endonuclease that causes an incision or nick on hemi-methylated DNA near the mismatch site

E. coli mismatch repair step 4

gap is filled in with DNA pol III and ligase

How are dsDNA breaks repaired

Nonhomologous end joining and homologous recombination are two primary mechanisms for repairing double-strand breaks in DNA.

Nonhomologous end joining step 1

Ku70 and Ku80 will recognize and bind to ends of the
dsDNA break and stabilize the ends

Nonhomologous end joining step 2

A protein complex of artemis and a DNA protein kinase
(DNA-PKcs) binds to Ku70 and Ku80

Nonhomologous end joining step 3

Artemis processes the broken ends to ensure they are
blunt-ended (potential for insertions or deletions)

Nonhomologous end joining step 4

ends are joined via ligase IV activity