Unit 4: DNA Repair Mechanisms and Homologous Recombination - Subjective Questions

Direct repair is a DNA repair mechanism in which the damaged nucleotide is directly reversed to its original form without removing the base or nucleotide and without the need for a template strand.

Photoreactivation by DNA Photolyase:

• UV radiation causes the formation of cyclobutane pyrimidine dimers (CPDs) between adjacent pyrimidines (usually thymines).
• DNA photolyase is the enzyme responsible for reversing this damage.
• The enzyme contains two chromophore cofactors:
  • FADH⁻ (reduced flavin adenine dinucleotide) – the catalytic cofactor.
  • A light-harvesting cofactor (either MTHF – methenyltetrahydrofolate, or 8-HDF – 8-hydroxy-5-deazaflavin).

Mechanism steps:

1. Photolyase binds to the pyrimidine dimer in a light-independent manner.
2. The light-harvesting cofactor absorbs a blue light photon (300–500 nm) and transfers the excitation energy to FADH⁻.
3. Excited **FADH⁻*** donates an electron to the cyclobutane ring of the dimer.
4. This causes the cyclobutane ring to break, restoring the two normal pyrimidine bases.
5. The electron is transferred back to the flavin, regenerating FADH⁻.

This process is called photoreactivation because it requires light energy for catalysis.

O⁶-methylguanine-DNA methyltransferase (MGMT), also called alkyltransferase, is a key direct repair enzyme that removes alkyl groups from the O⁶ position of guanine.

Background:

• Alkylating agents can add methyl or ethyl groups to the O⁶ position of guanine, creating O⁶-methylguanine.
• This lesion is highly mutagenic because O⁶-methylguanine pairs with thymine instead of cytosine, causing G:C → A:T transition mutations.

Mechanism:

• MGMT transfers the alkyl group directly from the O⁶ position of guanine to a cysteine residue in its own active site.
• The reaction restores the normal guanine base.

Key features (Suicide enzyme):

• MGMT is a "suicide" enzyme or single-use enzyme because the transfer of the alkyl group to its cysteine residue is irreversible.
• Once alkylated, the enzyme is permanently inactivated and targeted for degradation.
• Stoichiometry is 1:1 – one enzyme molecule repairs only one lesion.

Significance:

• MGMT protects cells against alkylation-induced carcinogenesis and mutagenesis.
• It is clinically relevant in cancer therapy, as high MGMT levels confer resistance to alkylating chemotherapeutic drugs.

Base Excision Repair (BER) corrects damage to single bases caused by oxidation, deamination, or alkylation without gross distortion of the DNA helix.

Steps of BER:

1. Recognition and base removal:

   • A specific DNA glycosylase recognizes the damaged/inappropriate base.
   • It cleaves the N-glycosidic bond between the base and the deoxyribose sugar, creating an AP site (apurinic/apyrimidinic site).

2. AP site cleavage:

   • AP endonuclease (APE1) nicks the phosphodiester backbone at the 5' side of the AP site, leaving a 3'-OH and a 5'-deoxyribose phosphate (dRP).

3. End processing:

   • dRP lyase activity (often part of DNA polymerase β) removes the 5'-dRP residue.

4. Gap filling:

   • DNA polymerase β (in short-patch repair) inserts the correct nucleotide.

5. Ligation:

   • DNA ligase III (with XRCC1) seals the nick, completing repair.

Types of DNA glycosylases:

• Monofunctional glycosylases – only remove the base (e.g., uracil DNA glycosylase, UNG).
• Bifunctional glycosylases – remove the base AND have AP lyase activity (e.g., OGG1).

Examples of repaired lesions:

• Uracil (from cytosine deamination)
• 8-oxoguanine (from oxidation)
• 3-methyladenine (from alkylation)

Nucleotide Excision Repair (NER) removes bulky, helix-distorting lesions such as pyrimidine dimers and chemical adducts by excising a short oligonucleotide containing the damage.

General steps of NER (in eukaryotes):

1. Damage recognition – The distorted region is recognized by repair proteins.
2. Helix opening – The TFIIH complex (with helicases XPB and XPD) unwinds ~25–30 bp around the lesion.
3. Dual incision – Endonucleases XPF-ERCC1 (5' side) and XPG (3' side) cut the damaged strand, releasing a ~24–32 nucleotide fragment.
4. Gap filling – DNA polymerase δ/ε synthesizes new DNA using the intact strand as template.
5. Ligation – DNA ligase I seals the nick.

Distinction between subpathways:

Clinical significance:

• Defects in NER cause Xeroderma Pigmentosum (XP), characterized by extreme UV sensitivity and skin cancer predisposition.
• Defects in TC-NER cause Cockayne Syndrome.

Mismatch Repair (MMR) corrects base-base mismatches and small insertion/deletion loops that escape the proofreading activity of DNA polymerase during replication.

Key proteins in E. coli:

• MutS – recognizes and binds mismatches.
• MutL – acts as a coordinator/molecular matchmaker.
• MutH – an endonuclease that nicks the newly synthesized strand.

Strand discrimination (crucial concept):

• In E. coli, the parental (template) strand is methylated at adenine residues in GATC sequences by Dam methylase.
• Immediately after replication, the new daughter strand is transiently unmethylated (hemimethylated state).
• MMR uses this hemimethylation to identify the unmethylated new strand as the one containing the error to be corrected.

Mechanism steps:

1. MutS recognizes and binds the mismatch.
2. MutL binds and links MutS to MutH.
3. MutH is activated and cleaves the unmethylated strand at a nearby GATC site.
4. Helicase II (UvrD) unwinds the DNA, and exonucleases degrade the strand from the nick past the mismatch.
5. DNA polymerase III resynthesizes the correct sequence.
6. DNA ligase seals the nick.

Significance:

• MMR increases replication fidelity ~100–1000 fold.
• In humans, defects in MMR genes (MSH2, MLH1) cause Hereditary Non-Polyposis Colorectal Cancer (HNPCC / Lynch syndrome).

Double-strand breaks (DSBs) are among the most dangerous DNA lesions, as they can lead to chromosomal translocations, deletions, and cell death. Two major pathways repair DSBs:

1. Non-Homologous End Joining (NHEJ):

• Directly ligates the broken DNA ends together.
• Key proteins: Ku70/Ku80 heterodimer, DNA-PKcs, Artemis, DNA ligase IV / XRCC4.
• Mechanism: Ku binds ends → recruits DNA-PKcs → ends processed → ligated.

2. Homologous Recombination (HR):

• Uses an undamaged homologous DNA sequence (sister chromatid) as a template.
• Key proteins: MRN complex, RAD51, RAD52, BRCA1/2.
• Mechanism: End resection → strand invasion → D-loop formation → repair synthesis → resolution.

Comparison Table:

Conclusion: NHEJ is quick but mutagenic, while HR is accurate but restricted to phases when a sister chromatid is available.

The Holliday Model, proposed by Robin Holliday in 1964, is a foundational model explaining the molecular mechanism of homologous recombination and genetic crossing-over.

Key Steps of the Holliday Model:

1. Alignment of homologous duplexes:

   • Two homologous DNA molecules align side by side.

2. Nicking of strands:

   • An endonuclease introduces single-strand nicks at corresponding sites in strands of the same polarity in both duplexes.

3. Strand exchange (invasion):

   • The nicked strands cross over and invade the homologous partner duplex, base-pairing with the complementary strand.

4. Ligation and Holliday junction formation:

   • DNA ligase seals the nicks, forming a cross-shaped structure called the Holliday junction (a four-stranded intermediate).

5. Branch migration:

   • The crossover point moves along the DNA, extending the region of heteroduplex DNA (strands from different parental molecules).

6. Resolution of the Holliday junction:

   • The junction is cut by resolvases. Depending on the orientation of cleavage, two outcomes arise:
     • Horizontal (patch/non-crossover) cut → Non-recombinant (spliced) products with only patches of heteroduplex.
     • Vertical (splice/crossover) cut → Recombinant (crossover) products with exchange of flanking markers.

Significance:

• The Holliday junction is a central intermediate in recombination.
• The model explains gene conversion and crossing over.

(The Holliday junction can be visualized as a chi (χ) structure under electron microscopy.)

The RecBCD pathway is the major pathway for homologous recombination and DSB repair in E. coli.

RecBCD Enzyme:

• A multifunctional enzyme complex with three subunits: RecB, RecC, and RecD.
• Possesses both helicase and nuclease (exonuclease) activities.

Steps of the RecBCD Pathway:

1. Entry and unwinding:

   • RecBCD binds to a blunt or nearly blunt double-strand end (e.g., a DSB).
   • It unwinds the DNA using ATP-dependent helicase activity, degrading both strands (with the 3'→5' strand degraded more vigorously).

2. Recognition of Chi (χ) site:

   • Chi sites are specific 8-nucleotide sequences: 5'-GCTGGTGG-3'.
   • These sites are recombination hotspots, occurring frequently in the E. coli genome.
   • When RecBCD encounters a Chi site, its nuclease activity is attenuated/modified.

3. Generation of 3' single-stranded tail:

   • After passing Chi, RecBCD stops degrading the 3'-ending strand, generating a 3' single-stranded overhang with the Chi sequence.

4. Loading of RecA:

   • RecBCD facilitates the loading of RecA protein onto the 3' ssDNA tail, forming a nucleoprotein filament.

5. Strand invasion (RecA function):

   • RecA promotes homologous pairing and strand invasion into a homologous duplex, forming a D-loop.
   • This leads to the formation of a Holliday junction.

6. Resolution:

   • The RuvABC complex processes and resolves the Holliday junction (RuvA/RuvB drive branch migration; RuvC is the resolvase).

Significance:

• Chi sites regulate where recombination is initiated.
• RecA is the central recombinase, homologous to eukaryotic RAD51.

Homologous recombination and site-specific recombination are two distinct mechanisms of DNA rearrangement.

Comparison Table:

Summary:

• Homologous recombination relies on sequence similarity and is important for repair and genetic variation.
• Site-specific recombination relies on specific recognition sequences and specialized enzymes, enabling precise DNA rearrangements.

The Cre/LoxP system is a site-specific recombination system derived from the bacteriophage P1, widely used as a genetic engineering tool.

Components:

1. Cre recombinase:

   • A 38 kDa tyrosine recombinase enzyme.
   • Belongs to the integrase family.
   • Does not require any cofactors or accessory proteins.

2. LoxP site (Locus of X-over of P1):

   • A 34 bp DNA sequence consisting of:
     • Two 13 bp palindromic (inverted repeat) arms.
     • A central 8 bp asymmetric spacer region.
   • The asymmetric spacer gives the LoxP site directionality/orientation.

Structure representation:

13 bp inverted repeat — 8 bp spacer — 13 bp inverted repeat
ATAACTTCGTATA - ATGTATGC - TATACGAAGTTAT

Mechanism of Cre-mediated recombination:

1. Four Cre monomers bind to the two LoxP sites (two Cre per LoxP).
2. The Cre proteins bring the two LoxP sites together, forming a synaptic complex.
3. Cre uses a catalytic tyrosine residue to cleave the DNA, forming a 3'-phosphotyrosine covalent intermediate.
4. Strand exchange occurs, generating a Holliday junction intermediate.
5. Resolution completes the recombination.

Outcomes depend on LoxP orientation:

• Same orientation (direct repeats) → Excision/deletion of intervening DNA.
• Opposite orientation (inverted repeats) → Inversion of intervening DNA.
• On separate molecules → Translocation/integration.

Applications:

• Conditional gene knockouts, tissue-specific gene deletion, and lineage tracing in mice.

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows the replication machinery to bypass lesions that would otherwise block replication, rather than repairing the damage itself.

Why TLS is needed:

• High-fidelity replicative polymerases (Pol III in bacteria, Pol δ/ε in eukaryotes) have tight active sites and cannot accommodate bulky or distorted lesions, causing replication fork stalling.

Mechanism:

1. When the replicative polymerase stalls at a lesion, it is temporarily replaced by a specialized TLS polymerase.
2. The TLS polymerase has a larger, more open active site that can accommodate damaged bases.
3. It inserts nucleotide(s) opposite the lesion, allowing replication to continue.
4. The high-fidelity polymerase then resumes normal replication (polymerase switching).

TLS Polymerases (Y-family):

• Pol η (eta) – bypasses UV-induced thymine dimers relatively accurately.
• Pol ι (iota), Pol κ (kappa) – bypass various lesions.
• Pol V (UmuD'₂C) and Pol IV (DinB) in E. coli (part of SOS response).

Why it is error-prone:

• TLS polymerases lack 3'→5' proofreading exonuclease activity.
• Their open active sites reduce base-selection fidelity.
• They may insert incorrect nucleotides opposite damaged/non-informative lesions.

Significance:

• TLS promotes cell survival at the cost of increased mutagenesis.
• It is a major source of induced mutations following DNA damage.

The SOS response is an inducible cellular response in E. coli activated by extensive DNA damage. It coordinates the expression of numerous genes involved in DNA repair and damage tolerance.

Key regulatory proteins:

1. LexA – the master repressor:

   • Under normal conditions, LexA binds to operator sequences (SOS boxes) and represses ~40 SOS genes.

2. RecA – the sensor and coprotease:

   • When DNA is damaged, single-stranded DNA (ssDNA) accumulates at stalled replication forks.
   • RecA binds this ssDNA, forming an activated *RecA nucleoprotein filament**.

Mechanism of SOS induction:

1. Activated **RecA* stimulates the autocatalytic self-cleavage** of LexA (RecA acts as a coprotease).
2. Cleaved LexA can no longer bind operators, derepressing SOS genes.
3. SOS genes are expressed, including:
   • uvrA, uvrB (NER)
   • recA (amplification)
   • sulA (cell division inhibitor – halts division to allow repair)
   • umuC, umuD (translesion synthesis / Pol V)

Recovery:

• Once damage is repaired, ssDNA disappears, RecA* is deactivated.
• LexA levels rise again, repressing SOS genes and restoring normal state.

Significance:

• The SOS response is a graded response – more damage leads to stronger induction.
• It enables both accurate repair and error-prone survival (mutagenesis).

The Double-Strand Break Repair (DSBR) model, proposed by Szostak, Orr-Weaver, Rothstein, and Stahl in 1983, refined the Holliday model to explain recombination initiated by double-strand breaks.

Steps of the DSBR model:

1. Double-strand break formation:

   • Recombination is initiated by a DSB in one DNA duplex (unlike the Holliday model that starts with single-strand nicks).

2. 5' end resection:

   • Exonucleases degrade the 5' ends, generating 3' single-stranded overhangs.

3. Strand invasion:

   • One 3' overhang invades the homologous intact duplex, displacing a strand and forming a D-loop.

4. DNA synthesis and D-loop expansion:

   • Repair synthesis extends the invading strand; the D-loop enlarges until the displaced strand pairs with the other 3' overhang.

5. Second-end capture and synthesis:

   • The second 3' end is captured and DNA synthesis fills the gaps.

6. Formation of TWO Holliday junctions:

   • Ligation produces a double Holliday junction intermediate (a key difference).

7. Resolution:

   • The two junctions are resolved by resolvases, yielding crossover or non-crossover products depending on cleavage orientation.

Differences from the Holliday Model:

Significance: The DSBR model better explains gene conversion and recombination during meiosis and DSB repair.

In E. coli, the processing and resolution of the Holliday junction is carried out by the RuvABC proteins, which act after RecA-mediated strand exchange.

1. RuvA:

• Function: Recognizes and binds specifically to the Holliday junction.
• Forms a tetramer that binds the crossover point, holding the junction in an open, square-planar configuration.
• Acts as a specificity/loading factor to recruit RuvB.

2. RuvB:

• Function: An ATP-dependent motor protein (helicase).
• Forms hexameric rings that assemble around the DNA arms on opposite sides of the junction.
• Uses ATP hydrolysis to drive branch migration, pumping DNA through the junction and extending the heteroduplex region.
• RuvA + RuvB together form the RuvAB branch migration complex.

3. RuvC:

• Function: The resolvase (endonuclease).
• Forms a dimer that binds the Holliday junction.
• Introduces symmetric cuts in two strands of like polarity at consensus sequences (5'-A/TTT↓G/C-3').
• Resolution can occur in two orientations:
  • One yields crossover (recombinant) products.
  • The other yields non-crossover (patch) products.

Overall sequence: RuvA binds junction → RuvB drives branch migration → RuvC cleaves and resolves the junction.

Significance: The RuvABC system is essential for completing recombination and DSB repair in bacteria.

Gene conversion is a non-reciprocal transfer of genetic information from one DNA molecule (donor) to a homologous DNA molecule (recipient), resulting in one allele being 'converted' into another. It violates the normal Mendelian expectation of a 2:2 segregation of alleles, often producing 3:1 ratios in fungal tetrads.

How gene conversion arises:

1. Heteroduplex DNA formation:

   • During homologous recombination, strand exchange creates a region of heteroduplex DNA, where one strand comes from each parental molecule.
   • If the two parental alleles differ at a site, this creates a base-pair mismatch within the heteroduplex.

2. Mismatch repair correction:

   • The cell's mismatch repair (MMR) system detects the mismatch.
   • Repair uses one strand as the template and corrects the other.
   • If repair uses the donor strand as template, the recipient allele is converted to the donor sequence.

Two mechanisms leading to gene conversion:

• Mismatch repair of heteroduplex DNA (as above).
• DNA synthesis during DSB repair – gaps are filled using the homologous donor as template, copying donor information.

Consequences:

• Produces aberrant segregation ratios (e.g., 6:2 or 5:3 in octads).
• An important mechanism for concerted evolution of gene families and generation of diversity (e.g., immunoglobulin genes).

Summary: Gene conversion results from the non-reciprocal 'copying' of genetic information during recombination, mediated primarily by heteroduplex formation and mismatch repair.

The integration of bacteriophage λ into the E. coli chromosome is a classic example of conserved site-specific recombination, mediated by the tyrosine recombinase family.

Key components:

1. att sites (attachment sites):

   • attP – on the phage genome (POP').
   • attB – on the bacterial chromosome (BOB').
   • Both share a common core (O) region where recombination occurs.
   • After integration, new hybrid sites attL (BOP') and attR (POB') are formed.

2. λ Integrase (Int):

   • A tyrosine recombinase encoded by the phage.
   • Binds the att sites and catalyzes strand cleavage and exchange via a covalent 3'-phosphotyrosine intermediate.

3. Integration Host Factor (IHF):

   • A bacterial DNA-bending protein.
   • Binds attP and bends DNA sharply, facilitating the assembly of the higher-order nucleoprotein complex called the intasome.

Mechanism of integration:

1. Int and IHF bind attP, forming the intasome.
2. The intasome captures attB.
3. Int makes staggered cuts in the core region, exchanges the first pair of strands (forming a Holliday junction), then the second pair.
4. Recombination results in the integration of the circular λ DNA into the bacterial chromosome, generating attL and attR.

Excision (reverse reaction):

• Requires Int, IHF, and Xis (excisionase).
• Recombination between attL and attR regenerates attP and attB, releasing the phage.

Significance:

• Demonstrates directionality control – integration vs. excision is regulated by accessory proteins (Xis).

Base Excision Repair (BER) can proceed via two subpathways that differ in the number of nucleotides replaced: short-patch and long-patch BER.

Common initial steps:

• A DNA glycosylase removes the damaged base, creating an AP site.
• AP endonuclease (APE1) cleaves the backbone, generating a 3'-OH and a 5'-dRP.

1. Short-Patch BER (single nucleotide replacement):

• Only ONE nucleotide is replaced.
• DNA polymerase β removes the 5'-dRP (via its dRP lyase activity) and inserts a single nucleotide.
• DNA ligase III / XRCC1 seals the nick.
• This is the predominant (major) pathway in mammalian cells.

2. Long-Patch BER (2–13 nucleotide replacement):

• 2 to ~13 nucleotides are replaced.
• DNA polymerase δ/ε (with PCNA) performs strand-displacement synthesis, creating a 5' flap.
• The flap is removed by FEN1 (flap endonuclease 1).
• DNA ligase I seals the nick.

Conditions determining pathway choice:

• Short-patch is used when the 5'-dRP is easily removed by Pol β.
• Long-patch is used when the 5' end is oxidized or reduced (modified) and resistant to Pol β's lyase activity.
• ATP levels and cell cycle stage can also influence pathway selection.

Summary: Both pathways restore correct sequence, but they differ in the polymerase, ligase, and number of nucleotides replaced.

Site-specific recombinases are broadly classified into two families based on their catalytic residue and mechanism: the tyrosine recombinases and the serine recombinases.

Comparison Table:

Tyrosine recombinase mechanism (conserved):

1. Tyrosine attacks the phosphodiester backbone, forming a 3'-phosphotyrosine bond.
2. One pair of strands is exchanged → Holliday junction forms.
3. The second pair of strands is exchanged → resolution.

Serine recombinase mechanism:

1. All four DNA strands are cleaved simultaneously, forming 5'-phosphoserine bonds.
2. Two halves of the complex rotate 180°.
3. Strands are religated in the recombinant configuration.

Significance:

• Both enable precise DNA rearrangements (integration, excision, inversion).
• Tyrosine recombinases like Cre and FLP are widely used genome-engineering tools.

The MRN complex is a central protein complex that senses and initiates the repair of double-strand breaks (DSBs), directing them toward homologous recombination (HR).

Components of the MRN complex:

• Mre11 – possesses nuclease activity (both 3'→5' exonuclease and endonuclease).
• Rad50 – an ATPase with long coiled-coil domains that tether DNA ends together.
• Nbs1 (Nibrin) – a regulatory subunit that recruits and activates the ATM kinase.

Roles of the MRN complex:

1. DSB sensing:

   • MRN is one of the first complexes to recognize and bind the broken DNA ends.

2. DNA end tethering:

   • Rad50 coiled-coils bridge the two broken ends, keeping them in proximity.

3. Signaling activation:

   • Nbs1 recruits ATM kinase, which phosphorylates H2AX (γH2AX) and other targets, amplifying the DNA damage response.

4. Initiation of end resection:

   • Together with CtIP, the MRN complex initiates 5'→3' end resection.
   • Mre11's nuclease creates a nick; further resection is carried out by EXO1 and DNA2/BLM.
   • This generates 3' single-stranded DNA (ssDNA) overhangs.

Downstream events:

• The 3' ssDNA is coated by RPA, which is then replaced by RAD51 (aided by BRCA2).
• The RAD51 nucleoprotein filament performs strand invasion into a homologous template.

Significance:

• End resection commits the DSB to HR rather than NHEJ.
• It occurs mainly in S/G2 phase when a sister chromatid is available.
• Defects in MRN components cause disorders like Nijmegen breakage syndrome and ataxia-telangiectasia-like disorder.

The Cre/LoxP system is one of the most powerful and versatile tools in modern genetic engineering, enabling precise, controllable manipulation of genomes.

Principle recap:

• Cre recombinase recognizes 34 bp LoxP sites and catalyzes recombination between them.
• Outcome (deletion, inversion, translocation) depends on LoxP orientation.

Major Applications:

1. Conditional (Tissue-Specific) Knockouts:

   • A gene of interest is "floxed" (flanked by two LoxP sites in the same orientation).
   • When Cre is expressed, the floxed gene is excised (deleted).
   • By placing Cre expression under a tissue-specific promoter, the gene is knocked out only in specific tissues/cell types, while remaining functional elsewhere.
   • This overcomes the problem of embryonic lethality seen in conventional whole-body knockouts.

2. Temporal (Inducible) Control:

   • Using CreER (Cre fused to a modified estrogen receptor), Cre activity is triggered only upon tamoxifen administration, allowing control over when the gene is deleted.

3. Lineage Tracing:

   • Cre activates a reporter gene (e.g., by removing a LoxP-flanked stop cassette) to permanently mark cells and their descendants.

4. Removal of Selection Markers:

   • Antibiotic-resistance cassettes flanked by LoxP sites can be excised after selection.

5. Chromosomal Engineering:

   • Generating targeted deletions, inversions, and translocations.

Concept of Conditional Knockout (summary):

• Requires two transgenic mouse lines:
  • One carrying the floxed target gene.
  • One expressing Cre under a tissue-specific promoter.
• Crossing them produces offspring where the gene is deleted only in the Cre-expressing tissue.

Significance:

• Enables the study of gene function in specific tissues, developmental stages, and disease models with high precision.