DNA Repair: Mechanistic Logic and Model Organisms

What are the two broad types of DNA damage?

Spontaneous and induced damage

What is spontaneous DNA damage?

Damage that occurs naturally from normal cellular chemistry

What is induced DNA damage?

Damage causes by environmental factors or cell stress

What are examples of spontaneous DNA damage

1. Depurination

2. Deamination

3. DNA base oxidation

4. Base alkylation

What is depurination?

The loss of a purine group (A or G) from DNA

What is deamination?

Removal of an amino acid group from a DNA base C —> U

What can cause DNA base oxidation?

ROS can damage a DNA base

What is base alkylation?

The addition of an alkyl group to a DNA base

What are types of induced DNA damage

1. UV Light

2. Bulky DNA Adducts

3. Ionizing radiation and collapsed replication forks = dsDNA break

What type of DNA damage does UV light cause

The UV radiation can cause adjacent thymine to abnormally fuse together and make a thymine dimer

What type of damage does toxic chemicals and cigarettes cause

Environmental toxins, cigarettes, smoke, or chemical agents attach large chemical groups to DNA bases which cause bulky DNA adducts that distort the DNA double helix

What can cause dsDNA damage

High energy from ionizing radiation can sever dsDNA at the same time, while a collapsed replication fork can block replication machinery and cause a collapse and eventually dsDNA break

What does Base Excision Repair (BER) fix?

Small, non-bulky damaged bases, such as ssDNA break, oxidized, depurination, deaminated, or single alkylated bases.

What does Nucleotide Excision Repair (NER) fix?

Bulky DNA lesions, including UV-induced thymine dimers or bulky DNA adducts

What does Mismatch Repair (MMR) fix?

Base mismatches that escape DNA polymerase proofreading.

What does double-strand break repair fix?

Breaks affecting both strands of DNA. Like in ionizing radiation or collapsed replication forks

What does direct reversal repair do?

Directly reverses certain chemical modifications without removing the DNA segment. Like in base alkylation

What cancers are associated with defects in BER?

Breast, ovarian, and pancreatic cancers.

What cancers are associated with defective double-strand break repair?

Breast and ovarian cancers

What cancer risk is classically associated with defective NER?

Skin and breast cancer

What cancer results from defective mismatch repair?

Colorectal cancer

What cancer is associated with defects in direct reversal pathways?

Glioma

What is alkylation of DNA?

Addition of an alkyl group, such as a methyl group (CH₃), to a DNA base.

What does alkylation damage of guanine produce

O⁶-methylguanine (O⁶-mG)

How does O⁶-methylguanine affect base pairing?

It pairs Guanine with thymine (T) instead of normal partner, cytosine (C)

How does O⁶-methylguanine cause a permanent mutation?

It pairs with thymine during replication, and eventually converts the original G-C pair to A-T

What happens when cytosine is methylated?

It becomes 5-methylcytosine and then deaminated to thymine.

What mutation can result from deamination of 5-methylcytosine?

Instead of cytosine to guanine it becomes thymine to adenine

Why are CpG sites mutation hotspots?

The cytosines can become methylated and undergo deamination, producing thymine and increasing C-G —> T-A mutations

What type of DNA damage does MGMT or O⁶-methylguanine DNA methyltransferase repair?

O⁶-methylguanine caused by alkylation.

How does MGMT repair O⁶-methylguanine?

It transfers the methyl group from guanine to a cysteine on itself by not cutting instead directly reverses the chemical damage.

Why is MGMT called a “suicide enzyme”?

After accepting a methyl group, MGMT becomes permanently inactive.

How can cancer cells use MGMT promoter methylation to increase mutations?

When they methylate the MGMT promoter which transfer away the methyl group from G, it reduces the transcription production of MGMT. This allows for methylguanine damage to accumulate, increasing mutations, including mutations in p53.

What enzyme starts Base excision repair?

DNA glycosylate, it recognizes and removes the damaged base, and creates an AP site leaving the sugar-phosphate backbone alone

What is an AP site?

A DNA site missing its base; AP means apurinic/apyrimidinic.

What does AP endonuclease do?

It recognizes the empty AP site and cleaves (cuts) the phosphodiester bond on the 5’ but leaves the baseless sugar residue still hanging onto the DNA strand.

What does DNA polymerase β do during BER?

It removes the remaining sugar residue after AP endonulcease leaves and adds the correct nucleotide

What does DNA ligase III do during BER?

It seals the remaining nicks in the DNA backbone.

What protein complex recognizes bulky DNA damage in NER?

XPC-HR23B.

What do XPB and XPD do during NER?

They act as helicase and unwind the DNA around the lesion.

What proteins cut out the damaged DNA during NER?

XPF and XPG and remove around 24–32 nucleotides.

What happens after the damaged DNA is excised in Nucleotide excision repair (NER)?

DNA polymerase δ or ε fills the gap and Ligase fills the gap

What is global NER?

NER that searches the entire genome for bulky lesions.

What is transcription-coupled NER?

NER that repairs damage blocking active transcription.

What does RER repair?

Ribonucleotides accidentally incorporated into DNA.

What is the difference between dNTPs and rNTPs?

dNTPs build DNA; rNTPs build RNA.

Why are rNTPs a problem when incorporated into DNA?

They can destabilize the DNA backbone and cause damage.

What enzyme recognizes and initiates removal of rNTPs?

RNase H2.

What happens after RNase H2 cuts during Ribonucleotide excision repair?

DNA polymerase δ or ε replaces the incorrect section.

What is a flap during RER?

A displaced DNA segment created during resynthesis

Which enzymes remove the flap during RER?

FEN1 or EXO1.

What determines which Double Strand repair pathway the cell uses?

The cell pathway and whether there is a sister chromatid available if the cycle is at G1 DNA replication hasn’t started yet and therefore no sister chromatid so you choose non-homo or SSA. On the other side if the cycle is at S/G2 DNA replication has started and a sister chromatid is available so Homo can be used

Why is Homo recombination high-fidelity?

It uses an intact sister chromatid as a template.

Why is Non-Homo end joining more error-prone?

It joins broken ends without using a template.

Why can SSA cause DNA loss?

it purposefully chews back and cuts away the genetic material till it meets two complementary sequences to bridge a double-strand break.

What is the first step of Homo recombination?

Recognition of the double break by MRN complex with MRE11, RAD50, and NBS1

What does BRCA1 help with during Homo recombination?

It promotes resection of the broken DNA by chewing away the 5′ ends of the break to expose single-stranded 3′ tails, acting like open velcro that can scout and lock onto a matching sister chromatid blueprint.

What protein coats the 3′ single-stranded DNA during Homo recombination?

RAD51, it promotes homology search and strand invasion in which a broken DNA strand enters and pairs with matching DNA on the sister chromatid

What serves as the template during homo recombination?

The intact sister chromatid

What enzyme copies the missing DNA during HR?

DNA polymerase.

What is a Holliday junction?

A crossed shape DNA structure formed during HR when the broken DNA strand invades and pairs with the homo DNA of the sister strand

What is branch migration?

Movement of the Holliday junction along the DNA.

What happens during Holliday junction resolution?

The junctions are cut and the DNA molecules are separated and sealed.

What kind of repair does non-homo do

It repairs double-strand breaks without using a sister chromatid. It processes/modifies the broken DNA ends and directly ligates them back together.

Why is NHEJ considered error-prone?

DNA may be added or removed while processing the broken ends.

When can NHEJ function during the cell cycle?

Throughout the cell cycle, including G1, S, and G2.

What can happen if NHEJ joins the wrong DNA ends?

Chromosomal rearrangements or translocations can occur.

What protein promotes Non-homo?

53BP1, it protects DNA ends from extensive resection where 5’ is chewed away and 3’ end is left as SS hanging

What protein promotes HR?

BRCA1, It promotes DNA end resection needed for HR.

What is PARP's basic role in DNA repair?

It helps cells respond to and repair single-strand breaks.

What can happen if an unrepaired SSB is encountered during replication?

The replication fork can collapse and produce a double strand break

What happens to HR when BRCA function is lost?

HR becomes defective and eventually cell dealth

What is a PARP inhibitor?

A drug that inhibits PARP-dependent DNA damage repair, for example Olaparib.

What is synthetic lethality?

Loss of either pathway alone is survivable, but loss of both causes cell death.

Why can PARP inhibition be lethal to BRCA-deficient cells?

DNA damage accumulates, leading to cell death.

What is the purpose of translesion synthesis (TLS)

To bypass DNA lesions that stall normal DNA replication.

What happens when normal DNA polymerase encounters a DNA lesion?

DNA synthesis can stall at the lesion and the leading strand synthesis can stall leading to uncoupling in synthesis between lead and lag.

Does TLS necessarily repair the original DNA lesion?

No. It allows replication to bypass the lesion.

What are the two main ways a stalled fork can bypass a lesion?

Switching the template from parental to intact newly synthesized DNA or lesion avoidance through specialized TLS polymerase

What happens during lesion avoidance/template switching?

Intact newly synthesized DNA is used as a temporary template through pairing with the stalled strand through branch migration

What happens after the missing DNA is synthesized during lesion avoidance?

Branch migration reverses and the replication fork is restored.

What happens in the TLS polymerase pathway?

A specialized polymerase synthesizes DNA directly across the lesion and after bypassing the lesion normal high fidelity DNA replication resumes.

Why can TLS polymerases copy across damaged DNA?

Their active sites can accommodate distorted or damaged bases.

Why are many TLS polymerases error-prone?

They have lower fidelity and often lack 3′→5′ proofreading which can cause mutations

What is the major difference between Nucleotide excision repair (NER) and translesion DNA synthesis (TLS)?

NER removes DNA damage; TLS allows replication to bypass it.

What is the major difference between the two bypass methods?

Template switching avoids copying the lesion; TLS polymerase copies across it.

What is the DNA Damage Response (DDR)?

A system that detects DNA damage and coordinates cellular responses through signaling kinases ATM and ATR

What type of DNA damage primarily activates ATR?

Single-stranded DNA (ssDNA) associated with replication stress.

What type of DNA damage primarily activates ATM?

Double-strand breaks (DSBs).

During which cell-cycle phase is ATR especially important?

S phase.

What checkpoint is strongly associated with ATM signaling?

The G2/M checkpoint.

How do ATM and ATR transmit damage signals?

By phosphorylating downstream proteins.

What are the major outcomes of DDR activation?

DNA repair, cell-cycle arrest, senescence, or apoptosis.

What is the effect of certain heterozygous ATM mutations?

Increased susceptibility to cancer.

What disorder results from pathogenic mutations affecting homozygous ATM copies?

Neurological degeneration and increased cancer susceptibility. Ataxia-telangiectasia (A-T).

What is an example of a drug targeting DNA damage response pathways?

Olaparib, a PARP inhibitor. They increase DNA damage that BRCA-deficient cells cannot efficiently repair by HR.

What is synthetic lethality?

Two combined defects cause cell death, while either defect alone may be survivable.