week 6 DNA damage

what biological molecules can undergo repair upon damage

DNA only

what happens to other biologial molecules upon damage

simply degraded and recycled

difference between DNA damage and DNA mutation

• DNA damage is the initial alteration of normal DNA structure

• If the cell systems fail to repair DNA damage prior to DNA replication, the damage will become a DNA mutation

can cells recognize dna damage

yes

The cell can recognize DNA damage as potentially lethal and will attempt to repair it immediately

can the cell recognize dna mutation

no

remains as a permanent change in the DNA sequence

are dna mutations or dna damage hertiable

dna mutations

table for differences between dna damage and dna mutations

After DNA damage occurs, there are three possible outcomes

1. successfull dna Repair

2. unsuccessful dna repair

3. no dna repair

when does successful dna repair occur?

repair proteins correct the DNA damage before DNA replication

when does unsuccessful dna repair occur? (use the example of cytosine → uracil)

• If the damaged DNA molecule undergoes a round of replication

• 2 non identical DNA molecules produced (1 that uses damaged strand as template and 1 that uses normal strand as template)

• uracil in damaged one pairs with adenine

• cytosine in normal one pairs with guanine

• result = adenine mutation!

• repair proteins remove the uracil because they can recognise it and replace it with T (thymine) because of the incorrect A

when does No dna repair occur

after incorporttion of the mutation

Spontaneous, or endogenous, DNA damage encompasses

any DNA damage that arises due to the nature of the cellular environment

three key sources of endogenous DNA damage

depurination/depyrimidation, deamination and reactive oxygen species

Depurination and deamination occur because

DNA is not chemically inert, leading to occasional spontaneous chemical modifications of its components

reactive oxygen species can inadvertently be generated during

energy metabolism, causing damage to DNA.

Depurination or depyrimidination is the spontaneous hydrolysis of a nucleotide's ______________ bond

N-glycosidic

Cleavage of this bond therefore releases the base from the nucleotide, forming an

apurinic site or apyrimidinic site (AP site), more generally referred to as an abasic site

most common form of DNA damage in humans

formation of abasic sites via depurination or depyrimidination

depurination is more common than losing a pyrimidine base (depyrimidination).

During DNA replication, if DNA polymerase alpha, epsilon, or delta encounters an abasic site

replication will stop

To bypass the abasic site and complete DNA replication, a specialized ___________________________) must be used.

trans-lesion synthesis polymerase (TLS

When an abasic site is encountered, TLS does what

incorporates a random nucleotide in the daughter strand which then allows DNA polymerase to continue replicating DNA. This process is highly mutagenic

consequence due to abasic sites

cleavage of the DNA backbone

minor relatively: TLS error rate

cleavage of the DNA backbone occurs in two steps

cyclic form → linear form

b elimination

beta-elimination breaks

breaking the 3’ phosphodiester bond of the abasic site.

products:

• 3’ aldehyde-terminated product (comprising the sugar and the non- cleaved phosphate) and a

• 5’ phosphate attached to its adjacent sugar

which bases have an exocyclic amine groups prone to spontaneous deamination

Adenine, guanine, and cytosine

what does the amine group become in deamination

a carbonyl group

amine: hydrogen bond donor

carbonyl: hydrogen bond acceptor

How are reactive oxygen species generated?

• produced within the mitochondria during oxidative phosphorylation

• The final complex (IV) in the ETC normally uses electrons to reduce oxygen to water.

• But 0.2-2% of electrons leak out of the ETC before reaching this final step, and they bind to oxygen and form superoxide anions

Superoxide anions are ______________ to the mitochondrial membrane

impermeable

therefore restricted to the mitochondria

what convert superoxide anions into hydrogen peroxide

Superoxide dismutase (SOD)

hydrogen peroxide is _______ to the mitochondrial membrane

permeable

If hydrogen peroxide is fully reduced, it will form

water molecule

partial reduction of hydrogen peroxide will lead to the formation of

hydroxyl radical that has the potential to cause DNA damage

Generation of hydroxyl radicals.

oxidized form of guanine

8-oxo-guanine ( addition of carbonyl group at position 8 of guanine)

what conformation does 8-oxo-guanine favour and why

syn (whereas normally it would be anti since guanine is a purine)

• bulky carbonyl group in 8-oxo-G causes significant steric clashing with the ribose sugar

Watson-Crick base pairing occurs when nitrogenous bases adopt ___________ conformation

anti

base pairing syn vs anti 8oG

Hoogsteen base pairs with adenine, instead of the typical Watson-Crick pairs with cytsoine

Environmental, or exogenous, DNA damage encompasses

DNA damage that occurs due to interaction with physical agents outside the cell

two key sources responsible for exogenous DNA damage;

UV light and alkylating agents

UV damage occurs when ____________ bases absorb UV light

pyrimidine

2 possibilities of uv light damage

2 bonds between adjacent pyrimidines => a cyclobutene pyrimidine dimer (CPD)

1 bond between adjacent pyrimidines => a 6-4 photoproduct (6-4) PP

distance between normal stacked/adjacent nucleotides in a DNA strand is

3.4 Å.

pyrimidine dimer distance

1.5 Å, equivalent to the length of the C-C bond that links them together

Structural changes to the double helix due to the presence of a pyrimidine dimer

replicative enzymes reach the pyrimidine dimer, they will stall, and their enzymatic activities will come to a halt. To resume their synthetic processes, they require the action of

a unique trans-lesion synthesis (TLS) polymerase. This TLS prefers to insert AA opposite to any pyrimidine dimer

this corrective mechanism fails if the dimer

involves any other pyrimidine combination (CC, TC, CT).

Alkylating damage occurs when alkyl groups are transferred to the ____________ of a nitrogenous base

nitrogen or oxygen atoms

One common type of alkylating damage is

the methylation of O6 in guanine, forming O⁶-methyl-guanine

Methylation of guanine at the O6 position causes two key alterations in the nucleotide’s base pairing

• O6 loses ability to participate in bond

• N1 becomes a h bond acceptor

O⁶-methyl-guanine forms __________ hydrogen bonds with ______________

two hydrogen bonds with either cytosine or thymine via Hoogsteen base pairing

during DNA replication, there is no preference as to which pyrimidine will be chosen so 50% chance of mutation

three key repair pathways

direct repair, base excision repair, and nucleotide excision repair

the damaged base O6- methylguanine is repaired by the enzyme

MGMT

enzymatic process associated with MGMT occurs in three phases

Accessing the damaged base through interdigitation.
Repairing the damaged base through a single-step chemical reaction.
MGMT is degraded after repairing DNA damage.

interdigitation

1. O⁶-methyl-guanine is flipped out of the DNA helix

2. cytosine it formerly based paired with in the opposite strand becomes an ‘orphan nucleotide’.

3. MGMT’s key arginine residue extends into the DNA helix through the minor groove

4. arginine residue is able to form hydrogen bonds with the ‘orphan nucleotide’

5. which stabilizes the DNA double helix while the O⁶-methyl-guanine undergoes repair in the MGMT active site

In the active site of MGMT, the _______ atom of a key ________ residue binds to the methyl group of the O6-methyl-guanine

sulfur

cysteine

what type of bond is formed in the repair enzymes active site

irreversible S-methyl bond

proteins like MGMT are referred to as

suicide enzymes

the enzyme loses its functionality and must be degraded

The cell's ability to repair alkylation damage is directly linked to

the quantity of repair enzymes it produces

In BER, these damaged bases are removed from the DNA strand and replaced with a new nucleotide using _________ enzymes

multiple different enzymes

four key steps of BER

• Cleave N-glycosidic bond to remove the damaged base

• Nick the DNA backbone

• Removal of deoxyribose monophosphate

• Fill the gap with a new nucleotide

A specific ________ identifies the damaged base and cleaves its __________ bond

DNA glycosylase

cleaves its N-glycosidic bond to generate an AP site

Each DNA glycosylase can only recognize one specific type of damaged base

how does DNA glycosylase specificity enhance DNA repair capacity

allows the cell to increase the production of DNA glycosylases specific for frequently occurring forms of damage

how does DNA glycosylase specificity reduces the types of damage that can be repaired by BER

DNA glycosylases are unable to identify novel forms of DNA damage

Once the damaged base has been removed, a_______ makes a nick in the DNA backbone

generates:

5’AP endonuclease

generates a 3’OH and 5’deoxyribose monophosphate

__________ removes the deoxyribose monophosphate to produce a one nucleotide gap

dRP lyase

what 2 enzymes are used to Fill the gap with a new nucleotide

what does NER recognize

general distortions in the DNA helix structure, which are caused by larger base-modifications during DNA damage

does not specifically recognize minor modifications of damaged bases, such as depurination or deamination

The enzymatic process of NER occurs in four key steps

The protein complex _______ recognizes and binds to the DNA double helix at sites of structural distortion

XPC-HR23B

TFIIH is composed of two subunits

XPB and XPD

the undamaged strand will be protected by the single stranded binding protein

RPA

_______________nuclease binds 3’ of the loop and _____________binds 5’ of the loop

XPG + XPA

XPF

Since multiple nucleotides are excised, the _______ is used to fill in the gap.

entire replisome

mismatch repair