DNA Repair

What is the difference between induced vs spontaneous DNA errors?

- Induced: mutation due to external agent (physical or chemical) or mutagen damaging the DNA

- Spontaneous: due to natural processes within cells

What are some factors that introduce DNA errors?

- DNA replication

- Free Radicals

- Ionizing Radiation

What are the consequences of Mutations?

1. Deleterious -> cause diseases

2. Advantages -> provided survival advantage to external factor

- source of genetic variation driving evolutionary change in response to changing environments

What are the different types of mutations?

1. Point Mutation: change in single nucleotide

2. Trinucleotide Expansion: expanding of 3 nuclotides

3. Chromosomal Rearrangement: diff parts of chromosome connected to diff areas

What are the types and classifications of point mutations?

Types:

1. Transition Mutation

2. Transversion Mutation

Classification:

- Silent

- Missense

- Frameshift

- Nonsense

What is the difference between a transition and transversion mutation?

1. Transition: one pyrimidine is replaced by another pyrimidine or one purine replaced by another purine

- favoured by spontaneous mutations cus it maintains shape

2. Transversion: purine replaced by pyrimidine or vice versa

Key:

Pyrimidine: T & C

Purine: A & G

How do point mutations create error in DNA replication?

Once mutation occurs due to DNAs semi-conservative nature after the second round or replication their is a mutant and wild type DNA

Where can point mutations occur?

1. non-coding RNA seq, altering structure and function

2. non-coding gene related areas, affecting gene regulation

3. protein coding genes

How does redundancy in the genetic code affect mutations?

1. Silent Mutation: change in nucleotide does not change amino acid

- aka synonymous mutations

- can still effect abundance of protein produced

2. Missense: change in nucleotide changes amino acid

- non-synonymous mutations

3. Nonsense: leads to premature stop codon

- makes truncated protein -> shorter polypeptide fragment

4. Frameshift: shifts/alters base sequence by...

- Addition of nucleotide

- Deletion of nucleotide

- if 3 added doesn't change frame but changes protein produced

How do Trinucleotide Expansions occur?

1. Microsatellites: Short DNA sequences (such as CAG) that are repeated multiple times in a row.

2. Strand slippage: During DNA replication, the DNA strands can temporarily misalign because of these repeated sequences.

3. Expansion: The newly made DNA strand can form a loop, causing the DNA polymerase to copy some repeats more than once.

4. Result: Extra repeat units are added, increasing the number of trinucleotide repeats in the DNA.

What is the consequence of Trinucleotide Expansions?

consequences:

- inc genetic instability

- leads to secondary structures

- can lead to gain or loss of function

- causes diseases that can be inherited

- triplets in disease gene increases as inherited

What are some secondary structures that result from trinucleotide expansions?

1. repeat with mirror symmetry: triplex DNA

2. Inverted repeat: cruciform

3. G-rich sequences: G-quad

What are the 3 major classes of DNA damage?

1. single base changes

2. structural distortion

3. DNA backbone damage

What is a Mutagen?

any chemical or physical agent that causes inc in rate of mutation above spontaneous backround

What are single base changes?

Conversion -> mutagen changes base on nucleotide in a DNA sequence

- causes minor structural distortion so may not block replication or transcription

- instead change in base pairing can cause point mutation

What are some examples of conversions?

1. C to U: due to deamination (losss of amine group)

2. methylC to T: due to deamination

3. G to methylG: due to alkylation

4. G to oxoG: due to oxidation

What are some examples of mutagens?

Extrinsic:

- X-rays (oxidiser)

Intrinsic:

- free radicals from metabolic processes (oxidisers)

- replication mistakes

What are the causes of structural distortions?

1. Pyrimidine dimers - Adjacent pyrimidines become covalently bonded due to UV light.

2. Intercalating agents - Molecules insert between base pairs and disrupt base stacking.

3. Base analogs - Similar-looking bases are incorporated but may pair incorrectly.

4. DNA adducts - Chemicals (ex:cancer) become covalently attached to DNA.

5. Protein attachments - Bulky proteins attach to DNA bases and obstruct enzymes.

How do Abaisic sites cause DNA Backbone Damage?

- AP sites occur when a nitrogenous base is removed from DNA.

- This happens when the N-glycosidic bond between the base and deoxyribose sugar is broken.

- The sugar-phosphate backbone remains intact, but the base is missing.

- They are among the most frequent DNA lesions.

Simple terms:

- Normally, DNA has a base attached to each sugar in its backbone. At an AP site, the sugar remains, but its base is missing.

- Why is this a problem? DNA polymerase may not know which nucleotide to insert opposite the missing base, potentially causing a mutation during replication.

How many DNA polymerases are in mammalian cells?

17 total:

4 replicative:

1. pol alpha: primes DNA synthesis during rep n repair

2. Pol delta: replicates lagging strand duriing rep n repair

3. Pol epsilon: replicates leading strand during rep n repair

4. Pol gamme: mitochondiral DNA rep n repair

- high fidelity repair (have low error rates)

13 DNA repair polyermerases

- error prone allowing error of 1:10-1:1000 per base pair

What are the diff responses to DNA damage?

1. Damage bypass

2. Damage reversal

3. Damage removal

What are the different types damage bypass mechanisms?

1. DNA synthesis

- damage: Pyrimidine dimer or apurinic site

- Repair proteins: DNA polymerases IV and V & pol η (eta)

What is DNA Bypass?

- translesion synthesis occurs

- instead of repairing, DNA polymerases replicates through the lesion by inserting an incorrect base or skipping the base

How does DNA polymerase read through this error?

- have replication factories

- the replication fork stalls when it encounters the lesion

1. Polymerase switching: at lesion, replicative polymerase is displaced and PCNS is loaded w a polymerase with help of REV1

2. Gap filling: replication machinery leaves ssDNA opposite the lesion, to be filled in by polymerase later

How is the lesion filled?

- synthesis is typically an error prone pathway

Prokaryotes:

- DNA Pol IV and Pol V participate in translesion synthesis in E. coli.

- Their activity is regulated by the SOS response.

- The SOS response activates genes involved in DNA damage tolerance and repair.

- DNA Pol IV is involved in bypassing certain lesions, while DNA Pol V can bypass a broader range of replication-blocking lesions.

- DNA Pol IV and Pol V are error-prone, so their activity can increase the risk of mutations

Eukaryotes: high fidelity synthesis achieved by DNA pol η (eta)

What are the different types damage reversal mechanisms?

1. Photoreactivation

- damage: Pyrimidine dimers

- repair proteins: DNA photolyase

2. Removal of methyl groups

- damage: O6‐methylguanine

- repair proteins: Methyltransferase

3. Removal of DNA-protein cross-links

- damage: DNA-protein cross‐links

- repair proteins: SPRTN protease

What is damage reversal?

- done via distinct enzymes

In prokaryotes and some eukaryotes:

- damage: Pyrimidine-Pyrimidine Dimers caused by UVlight

- repaired by photoreactivation

In eukaryotes and prokaryotes:

- damage: reversal of base methylation, such as O6-methylguanine, is

- reverse by methyltransferases•

IN multicellular eukaryotes:

- damage DNA-protein crosslinks

- reversed by SPTRN.

What is Photoreactivation?

- Damage and agent: UV light damage results in formation of pyrimidine-pyrimidine dimers

Repaired by:

- DNA photolyases are enzymes that uses visible light (near-UV to blue light or 300nm-500nm light) to break the covalent bonds holding the adjacent pyrimidines together.

- They require two co-factors:

1. FADH- (electron transfer)

2. a chromophore (pigment)

Mechanism not present in placental mammals

What is Demethylation of O6-methylguanine?

- Damage and Agent: O6-methylation of guanine by alkylating agents

Repaired by:

- Function: Repairs O⁶-methylguanine by removing the damaging methyl group from guanine.

- Binds to DNA: Binds in the minor groove, widens the groove, and bends the DNA to flip the damaged base into the major groove for access.

- Active site: Contains a cysteine residue that accepts the methyl group from guanine.

- Terminal reaction: The methyl group is transferred permanently to MGMT's cysteine. MGMT is irreversibly inactivated and degraded after one repair reaction, so it is called a suicide enzyme.

What is removal of DNA protein crosslinks?

- Damage and Agent: DNA-protein crosslinks(DPCs) induced when proteins become and remain covalently attached to the nitrogenous bases, sugar or broken phosphodiester bond on the DNA backbone

Repaired by:•

- SPRTN, a multidomain protein, that associates with theDNA replication machinery. It is a DNA dependent metalloprotease.

- It degrades proteins associated with DNA, and DNA binding and the associated conformational change are essential for SPRTN activation

- Regulated by the Ubiquitin.

What are the different types damage removal mechanisms?

1. Base excision repair

- damage: Damaged base

- repair proteins: DNA glycosylases

2. Mismatch repair

- damage: Replication errors

- repair proteins: MutSα, MutLα, and EXO1 in humans

3. Nucleotide excision repair

- damage: Pyrimidine dimer Bulky adduct on base

- repair proteins: XPA, XPB, XPC, XPD,ERCC1/XPF, and XPG in humans

4. Double-strand break repair

- damage: Double‐strand breaks

- repair proteins:

- MRN complex, Rad51, Rad 52,BRCA1, BRCA2, XRCC3, etc. in humans for homologous recombination

- Ku proteins, Artemis/DNA‐PKCS, XRCC4 inhumans for nonhomologous end‐joining

What is damage removal?

- involves multiple proteins that interact w each other at diff stages

General overview:

1. DNA repair machinery must first gain access to the DNA

2. DNA repair machinery removes damge

3. gaps filled

4. histones redeposited

What is base excision repair (BER)?

Repairs small, damaged DNA bases by removing the damaged base and replacing it with the correct nucleotide.

What is the first step to BER?

Step 1: Recognition and removal of the damaged base

- DNA glycosylases recognize and remove damaged bases, creating an abasic (AP) site — a location where the base is missing from the sugar-phosphate backbone.

Examples:

- Uracil DNA glycosylase: Removes uracil from UA or UG base pairs.

- hOGG1: Removes 8-oxoguanine (oxoG), a damaged form of guanine.

- The abasic site must then be repaired.

How does short patch repair work for BER?

1. APE1: Cuts the DNA backbone on the 5′ side of the AP site, leaving a 3′-OH and a 5′-dRP group.

2. DNA polymerase β (Pol β):

- Uses its β-lyase activity to remove the 5′-dRP group.

- Inserts the correct replacement nucleotide by extending from the 3′-OH end.

3. DNA ligase III-XRCC1 complex: Seals the remaining nick, completing repair.

How does long patch repair work for BER?

1. APE1: Makes a cut (nick) at the 5′ side of the abasic site.

2. DNA polymerase δ or ε + PCNA: Synthesizes replacement

3. DNA, displacing the existing strand to create a flap of 2-10 nucleotides.

4. FEN1: Cuts off the displaced DNA flap.

5. DNA ligase I: Seals the backbone, completing repair.

What is mismatch repair (MMR)?

Corrects mismatched DNA base pairs that escape proofreading during DNA replication.

What is the first step to MMR?

Step 1: Mismatch recognition and strand identification

- The mismatch must be recognized, and the cell must identify which strand is newly synthesized and contains the error.

- In E. coli: The parental strand is methylated at GATC sites, while the newly synthesized strand is temporarily unmethylated. The unmethylated strand is identified as the one to repair, and a nick is made in it.

- In eukaryotes: Strand identification involves signals such as nicks in the newly synthesized strand and interactions with PCNA; it is more complex than the bacterial system.

What is the second step to MMR?

Step 2: MSH sliding clamps are loaded

- MSH proteins recognize the mismatch and form ATP-bound sliding clamps.

- These clamps help recruit and coordinate repair proteins, including EXO1, which removes the mismatched section.

What is the third step to MMR?

- The DNA is cut and removed from either the 5′ or 3′ side of the mismatch, depending on the location of the strand nick.

- Differs between 5' end and 3' end

What happens at the 5' end in MMR?

1. An ATP-bound MSH clamp helps hold EXO1 at the 5′ nick.

2. EXO1 moves 5′ → 3′, removing DNA from the damaged strand, including the mismatch.

3. This may remove several thousand nucleotides.

4. RPA coats the exposed single-stranded DNA (ssDNA) to protect it.

- Additional MSH sliding clamps can help recruit EXO1 to continue excision.

What happens at the 3' end in MMR?

1. PCNA is associated with the 3′ nick.

2. An ATP-bound MSH clamp associates with MLH/PMS, and the complex moves along the DNA toward PCNA.

3. Interaction with PCNA activates the endonuclease activity of MLH/PMS.

4. MLH/PMS makes multiple single-strand cuts (nicks) in the DNA.

5. EXO1, a 5′ → 3′ exonuclease, removes DNA from these nicks until the mismatch is eliminated.

What is the fourth step of MMR?

Step 4: DNA resynthesis and sealing

- DNA polymerase δ: Synthesizes new DNA to replace the excised section, using the correct parental strand as a template.

- DNA ligase I: Seals the remaining nick by forming the final phosphodiester bond in the sugar-phosphate backbone.

What are the abbreviations, errors and agents to DNA repair methods?

1. Mismatch Repair (MMR)

- error: replication

2. Base Excision Repair (BER)

- error: damaged bases

- agents: oxidation, alkylation, deamination

3. Nucleotide Excision Repair (NER)

- error: bulky adducts

- agents: UV radiation, bulky chemical compounds

4. Homologous Recombination (HR)

- error: double strand breaks

- agents: ionizing radiation

5. Non-homologous End Rejoining (NHEJ)

- error: double strand breaks

What are three ways to respond to pyrimidine dimers

1. Bypass

- DNA polymerase copies across the damaged site so replication can continue. Usually error-prone.

- key protein: Translesion polymerases, e.g. Pol η

2. Reversal

- Directly reverses the dimer without removing the DNA section. Not available in all organisms.

- key protein: DNA photolyase

3. Removal

- Cuts out the damaged DNA section and replaces it with newly synthesized DNA.

- key protein: NER proteins

What is Xeroderma pigmentosum (XP)?

- Cause: Rare autosomal recessive genetic disorder.

- Problem: Cells cannot efficiently repair UV-induced DNA damage.

- Result: Extreme sensitivity to UV light and a greatly increased risk of skin cancer.

Genes involved:

- Seven XP-associated genes: XPA, XPB, XPC, XPD, XPE, XPF and XPG. These encode proteins involved in nucleotide excision repair.

- The eighth gene: Encodes DNA polymerase η (Pol η), which performs translesion synthesis.

Important distinction: Most XP-associated genes affect NER, while the eighth, POLH, affects translesion synthesis. A defect in Pol η prevents accurate bypass of UV-induced pyrimidine dimers.

What is the nucleotide excision repair pathway (NER)?

NER removes a short section of DNA containing a bulky lesion, such as a pyrimidine dimer, and replaces it using the undamaged strand as a template.

What is the first step to NER?

Step 1: Recognize the damage

NER has two recognition pathways:

1. Global genome repair (GG-NER): Detects DNA damage throughout the genome, including regions not actively being transcribed.

2. Transcription-coupled repair (TC-NER): Detects damage when RNA polymerase stalls while transcribing an active gene.

What is the second step of NER?

Step 2: Global genome repair (GG-NER)

- XPC: Scans DNA for structural distortions. It is an indirect damage sensor because it recognizes abnormalities in DNA structure rather than identifying every damaged base directly.

- When XPC detects a possible lesion, it opens the DNA and flips out the damaged bases.

- XPA, RPA and TFIIH help verify the lesion and organize the repair machinery.

What is the third step of NER?

Step 3: Unwind the DNA

- TFIIH contains the XPB and XPD helicases.

- These proteins unwind the DNA around the damaged site, exposing the damaged strand so it can be removed.

What is the fourth step of NER?

Step 4: Cut out the damaged section

- Two endonucleases cut the damaged strand on opposite sides of the lesion:

- XPG: Cuts on the 3′ side of the damage.

- XPF-ERCC1: Cuts on the 5′ side of the damage.

- The damaged DNA segment is released.

What is the last step of NER?

Step 5: Fill and seal the gap

- DNA polymerase δ or ε: Synthesizes replacement DNA using the undamaged strand as the template.

- RFC: Loads PCNA onto DNA.

- PCNA: Helps DNA polymerase synthesize the replacement strand.

- RPA: Binds and protects exposed single-stranded DNA.

- DNA ligase: Seals the remaining nick to complete repair.

What is the difference between Global genome repair and transcription-coupled repair?

1. Global genome repair

- Where: Throughout the genome

- What detects damage: XPC recognizes structural distortions

- Main purpose: protect entire genome

2. Transcripton-coupled repair

- Where: Transcribed strand of active genes

- What detects damage: Stalled RNA polymerase signals damage

- Main purpose: Quickly restore transcription of active genes

What are the key proteins of NER?

- TFIIH (contains a helicase XPB and XPD) unwinds the DNA double helix

- XPG an endonuclease nicks the DNA 3' of the damage and endonuclease XPF nicks the DNA 5' of the damage.

- TFIIH (contains a helicase XPB and XPD) unwinds the DNA double helix and allows for the damage strand to be released.

- Repair completed by DNA Pole or Pold RFC, RPA and PCNA

How does mismatch repair happen when at the 5' end?

Step 1: MSH binds DNA

- MSH binds ATP and forms a sliding clamp.

- It holds EXO1 at the 5′ strand nick and helps it move in the 5′ → 3′ direction.

Step 2: EXO1 removes DNA

- EXO1 digests the damaged strand, removing the mismatch.

- It can remove thousands of nucleotides.

- RPA coats the exposed single-stranded DNA (ssDNA) to protect it.

- Additional MSH clamps can help EXO1 continue digestion.

How does mismatch repair happen when at the 3' end?

Step 1: Proteins assemble

- PCNA binds at the 3′ nick.

- ATP-bound MSH associates with MLH/PMS.

- This complex moves along DNA toward PCNA.

Step 2: More nicks are created

- Interaction with PCNA activates the MLH/PMS endonuclease.

- MLH/PMS creates multiple single-strand nicks in the 5′ direction from the 3′ end.

- EXO1 uses these nicks to remove the mismatched strand.

- The process continues until the mismatch is removed

What are the final steps of DNA repair?

1. DNA polymerase δ

- Synthesizes new DNA to replace the removed section using the correct strand as a template.

2. DNA ligase I

- Seals the remaining nick by forming the final phosphodiester bond.

What is a double strand break and how is it caused?

- when both strands of the DNA double helix break at or near the same location.

Causes:

- Reactive oxygen species (ROS): Reactive molecules that damage DNA.

- Ionizing radiation: Such as X-rays, which can break DNA strands.

- Chemicals: Certain chemicals generate ROS, which can damage DNA.

How are double-strand breaks repaired?

A. Homologous Recombination (HR)

- High fidelity: Usually repairs DNA accurately.

- Requires a template: Uses a matching DNA sequence, usually from a sister chromatid.

- When: Typically occurs during late S and G2 phases, when a sister chromatid is available.

- primary method in prok & lower euk

B. Non-Homologous End Joining (NHEJ)

- Error-prone: Can introduce small insertions or deletions.

- No template required: Directly joins the broken DNA ends.

- When: Can function throughout the cell cycle.

- primary method in somatic cells, higher euk like mammals

What are the steps to Non-Homologous End Joining (NHEJ)?

1. DNA breaks

- Both strands of the DNA break, creating two DNA ends that may slowly drift apart.

2. Ku70/Ku80 bind the ends

- The Ku heterodimer binds to the broken DNA ends.

- It recruits DNA-PKcs (DNA-dependent protein kinase catalytic subunit).

3. Synapse formation

- The broken ends are held close together so they can be repaired.

4. DNA end processing

- Nucleases, such as Artemis, remove damaged or incompatible DNA sections using exonuclease or endonuclease activity.

5. DNA polymerase fills gaps

- DNA polymerases add nucleotides to fill missing sections of DNA.

6. DNA ligase IV/XRCC4 seals the break

- The complex joins the DNA ends by forming phosphodiester bonds, restoring the DNA backbone.

What are the key proteins and their functions in NHEJ?

1. Ku70/Ku80 - Recognizes and binds broken DNA ends

2. DNA-PKcs - Helps coordinate DNA end repair

3. Artemis - Cuts and processes DNA ends

4. DNA polymerase - Fills gaps with nucleotides

5. DNA ligase IV/XRCC4 - Joins and seals the DNA ends

What is homologous recombination (HR)?

DNA repair mechanism that repairs double-strand breaks (DSBs) by using an undamaged, homologous DNA molecule as a template to restore the missing genetic information.

What are some key features of HR?

- Requires sequence homology: The undamaged DNA must contain a matching or highly similar DNA sequence.

- Template: Usually a sister chromatid, although a homologous chromosome can also provide the template.

- High fidelity: HR is generally accurate because it copies information from an undamaged DNA molecule.

- Strand exchange: DNA strands from separate DNA molecules interact and exchange pairing partners.

- Genetic diversity: HR also contributes to genetic variation during meiosis through crossing over

What is the key signal transducer of HR?

Key signal transducer: ATM

- ATM (ataxia-telangiectasia mutated) is a serine/threonine kinase.

- It is recruited following DNA damage.

- It phosphorylates proteins involved in the DNA damage response.

- This helps activate and coordinate DNA repair pathways, including HR.

What are the first two steps to HR?

Step 1: Initiation

- A double-strand break is detected.

- The MRN complex (MRE11, RAD50 and NBS1) binds to the broken DNA ends.

- MRN recruits and activates ATM, which phosphorylates proteins involved in the repair response.

Step 2: Resection

- Nucleotides are removed from the 5′ ends of the broken DNA.

- This produces exposed single-stranded 3′ tails.

- RPA (replication protein A) rapidly binds the single-stranded DNA to protect it.

What is the third step to HR?

Step 3: Strand invasion

- RAD51 replaces RPA and forms a nucleoprotein filament on the single-stranded DNA.

-BRCA1-PALB2 and BRCA2-DSS1 help facilitate RAD51 loading and RPA displacement.

- RAD51 promotes invasion of the undamaged homologous DNA molecule.

- The invading strand pairs with its complementary sequence and displaces the original strand, forming a D-loop (displacement loop).

What is the fourth step to HR?

Step 4: DNA synthesis

- The invading 3′ end acts as a primer for DNA synthesis.

- DNA polymerases copy genetic information from the undamaged template.

- PCNA helps DNA polymerases δ and ε synthesize the replacement DNA.

- Depending on the pathway, the newly synthesized strand can pair with the other broken DNA end, forming a heteroduplex and potentially a double Holliday junction.

What are the two final steps to HR?

Step 5: Branch migration

- The Holliday junction can move along the DNA.

- This changes the regions where the DNA strands are paired.

- It can extend the heteroduplex DNA region.

Step 6: Holliday junction resolution

- A Holliday junction (HJ) is a four-stranded DNA intermediate formed during recombination.

- Enzymes resolve or dissolve the junctions to separate the DNA molecules.

- The final products may be crossover or non-crossover products, depending on the pathway and processing mechanism.

What is the D-loop and its importance in HR?

The D-loop is formed when the invading 3′ single-stranded DNA pairs with a complementary sequence in the undamaged homologous DNA, displacing one of its original strands.

Why is this important?

- It allows the broken DNA to access an intact template.

- The invading 3′ end can be extended by DNA polymerase.

- The new DNA copies the missing genetic information from the undamaged template.