DNA Damage and Repair

• Living organisms are constantly exposed to DNA-damaging agents that affect health.

• DNA repair mechanisms protect DNA by removing or tolerating damage to ensure survival.

• Mutations need to be kept at an acceptable level.

• DNA repair minimizes cell killing, mutations, replication errors, DNA damage persistence, and genomic instability.

• Preserving genomic sequence information is crucial for life, and mutagenesis plays a role in maintenance, evolution, cancer, diseases, and aging.

• Cells respond to DNA damage through DNA damage response (DDR) pathways.

• These pathways allow time for specific DNA repair to remove the damage in a substrate-dependent manner.

• Genome maintenance requires coordination between replication and repair.

Environmental Mutagenic Agents and DNA Damage

• Environmental agents, such as UV radiations, chemical mutagens, fungal toxins, and ionizing radiations, cause DNA damage in cells.
• UV B radiation causes various DNA lesions.
• These lesions include cyclobutane pyrimidine dimers (CPDs), pyrimidine pyrimidinone dimers (PPDs), oxidized or hydrated bases, and single-strand breaks.

Effects of UV Radiation on DNA

• UV radiations are readily absorbed by purines and pyrimidines, making them more reactive.

• Even low-energy UV light can be mutagenic, especially for unicellular organisms.

• DNA’s maximum UV absorption occurs at 254 nm.

• Maximum mutagenicity also occurs at 254 nm.

• UV-induced mutations are mediated by the absorption of purines (pu) and pyrimidines (py).

• Thymine (T) strongly absorbs at 254 nm, forming intra-strand linkages called T dimers.

• Polymerases struggle to replicate past T dimers, causing stuttering.

• Cytosine (C) can be converted to cytosine hydrate.

Early Discoveries in DNA Repair

• DNA repair was first observed when bacteria showed increased survival after irradiation.

• This was an increase in their surviving fraction after being subjected to post-irradiation treatments

• Mutant strains more sensitive to radiation than wild-type strains were observed

• Repair of UV damage was observed as increases in survival when bacterial cells exposed to sunlight were left in a window before forming colonies (photoreactivation).

• Exposure to visible light eliminates some UV induced damages.

• "Liquid-holding recovery" is another response to DNA damage.

• Irradiated cells held in a non-nutrient buffer for several hours showed improved survival.

• Dark or light independent repair systems were identified from extraordinarily UV sensitive E. coli mutant called Bs, where Bs mutant showed no increased viability after UV radiation when held in buffer in the dark but does show photoreactivation normally.

Types of DNA Repair

• Dark repair reaction removes T dimers differently from light repair mechanisms.

• Dark repair completely eliminates T dimers.

• Light repair cleaves and converts T dimers to monomers.

• Cells have evolved UV damage repair systems even though UV radiation isn't common in nature, because of DNA repair

• There are three categories of repair mechanisms.

• Damage reversal: Enzymatic action restores the normal structure without breaking the backbone.

• Damage removal: This involves cutting out and replacing damaged bases or nucleotide sections.

• Damage tolerance: This coping mechanism does not truly repair the damage but allows life to continue.

• Most DNA repair mechanisms have been studied in E. coli.

Photoreactivation

• Pyrimidine dimer formation is lethal unless corrected via photoreactivation, a repair mechanism.

• When bacteria exposed to UV radiation are subsequently exposed to visible light (300-450 nm), survival rate increases and mutation frequency decreases.

• Photoreactivation is also called direct repair

• It involves an enzyme that splits the T dimer without removing nucleotides.

• The enzyme binds to the dimer in the dark and requires energy from visible light to catalyze cleavage.

• The enzyme binds to the T dimer along with its cofactor folic acid.

• When light shines on the cell, folic acid absorbs it, and the enzyme uses this energy to break dimer bonds.

• The photolyase enzyme falls off the DNA after the dimer is split.

• The enzyme is also active on C-C and C-T dimers.

• Up to 80% of thymine dimers can be photoreactivated.

Excision Repair System

• This system removes and replaces altered bases from damaged DNA.
• It handles UV damage and AP repair.

UV Damage Repair/Nucleotide Excision Repair (NER)

• NER involves cutting out and replacing defective nucleotides.

• Analysis of dark repair came from the study of UV-sensitive mutants.

• Complementation tests revealed three categories of mutations: uvrA, uvrB, and uvrC.

• Recombination deficient mutants were isolated in E. coli; these mutations mapped to recA, recB, and recC genes.

• rec mutants are UV sensitive, but rec genes are involved in another class of repair, indicating the rec mutants still can excise T dimers.

• The process involves enzyme-catalyzed steps where T dimers are removed and new strands are synthesized.

• NER occurs efficiently in the dark.

Protein Functions in NER

• Excinucleases (endonuclease and exonuclease activity) hydrolyze two phosphodiester bonds, creating 3'-OH and 5'-P groups on either side of the lesion.

• UvrA, UvrB, and UvrC proteins are involved; UvrC nicks the DNA 8 bases upstream and 4-5 bases downstream of the dimer.

• UvrD (DNA helicase) separates strands to release a 12 base pair segment.

• After an excised nucleotide is removed, the resulting gap is filled by DNA polymerase I in E. coli and DNA polymerase E in humans, then joined by DNA ligase.

Xeroderma Pigmentosum

• Xeroderma Pigmentosum disease results from a deficiency in a DNA repair enzyme (UV exinuclease).
• Patients lack UV endonuclease activity and cannot repair T dimers.
• They exhibit extreme photosensitivity.
• Skin becomes dry and atrophies, and tumors develop.
• Death by age 30 from skin cancer is common
• There's no treatment available; patients must avoid sunlight.

Nucleotide Excision Repair Steps

• UvrA recognizes bulky lesions, creating a structural distortion signal.
• UvrB and UvrC make cuts on either side of the lesion.
• UvrD (DNA helicase) removes the DNA segment. .
• DNA polymerase fills the gap using the intact strand as a template.
• DNA ligase seals the nick to restore the DNA strand.

AP Repair / Base Excision Repair

• Repairs apurinic or apyrimidinic sites where bases have been removed.

• Modified bases are recognized and cut out.

• Mutations are caused by alkylation and deamination.

• Damaged or modified bases are recognized by special DNA glycosylase enzymes.

• The ung gene codes for uracil-DNA glycosylase, which removes uracil from DNA by cleaving the sugar nitrogen bond.

• AP endonucleases class I nick the 3’ side of AP sites, and class II nick the 5’ side.

• Exonuclease removes a short region of DNA, DNA Pol I fills the gap, and ligase reseals it.

Mismatch Repair (MMR)

• Accounts for 99% of all DNA repairs.

• Follows behind the replication fork.

• Corrects mistakes made during replication via 3'-5' exonuclease proofreading and mismatch repair.

• Requires mutH, mutL, mutS, and mutU gene products (mutator genes).

• The system recognizes progeny strands rather than parent strands to correct mismatches.

• DNA methylase (coded by dam (DNA adenine methylase)) methylates 5'-GATC-3' sequences in DNA at adenine (A).

• Mismatches are recognized by mutL and mutS gene products.

• NA helicase II, from the mutU gene (also called uvrD), unwinds the DNA duplex and releases the nicked region.

• Gaps are filled and sealed by DNA Pol I and ligase.

• Three proteins (MutS, MutL, and MutH) are required for the recognition of the mutation and nicking of the strand.

• Other enzymes, including ligase, polymerase and SSBs remove and replace the strand.

• Process is more complicated in mammalian cells, with about six proteins involved in the first steps.

• Faulty mismatch repair has been linked to hereditary nonpolyposis colon cancer (HNPCC), a common inherited cancer

Post-Replicative Recombination Repair

• It is a double-strand break (DSB) repair mechanism.

• It is a dark repair mechanism.

• Both replication and recombination are involved.

• If a DNA strand contains lesions that prevent base pairing, gaps are created in the daughter strand during replication.

• This causes breaks in both strands

• The gap is filled by sequence information from the parent strand of the sister chromosome via homologous recombination.

• The parent strand is repaired by DNA polymerase I.

• Sister chromosomes recombine such that dimers and gaps end up in the other chromosome, producing a viable cell.

• RecA protein (38 kD) binds to single-stranded DNA (s/s DNA) and carries out ATP-driven strand displacement (branch migration).

• BCD (328 kD) complex coding exonuclease V cuts for recombination at 5'-GCTGGTGG-3' (*chi) *specific sites occuring about every 4kb in E.coli.

• Chi sequences are crossover hotspot instigators

SOS Repair/Response

• Photoreactivation, excision repair, and post-reactive recombination repair are generally error-free.

• There also exists error-prone, mutation-inducing repair called SOS repair.

• SOS repair is also known as inducible repair.

• It involves over 40 genes responsible for DNA protection, replication, repair, and mutation.

• SOS response has been found in E. coli, Salmonella typhimurium, and Mycobacterium tuberculosis, but not in eukaryotic cells

• It is not a single mechanism but includes diverse responses to repair thymine dimers, induce prophages, stop respiration, and delay septum formation during cell division.

• All responses are coordinated.

• International Morse code distress signal: “save our souls.”

• SOS response occurs when cells are overwhelmed by UV damage and unrepaired DNA accumulates, allowing cells to survive at the cost of mutagenesis.

• Cells remain mutated but are able to survive.

• The SOS response is an error-prone, bypass system.

• Requires the function of the recA protein

• Normal function of the regulatory protein LexA is to repress the expression of approximately 20 genes involved in emergency repair processes

• Each genes features a consensus sequence in its promoter named the SOS box with the sequence 5’ CTGX10CAG3’ that serves as binding site for LexA protein

• The recA protein causes LexA stimulatation to break down autoregulatorily.

• The SOS response is induced when DNA is damaged or when replication stops and single stranded DNA accumulates

• Initiated by agents such as UV radiation, methyl methane sulphonate, as well as chemicals that cause DNA damage

• lexA encodes the lexA protein which is the repressor, binding to the SOS repressor box of the promoter sequence of the SOS gene expression.

• RecA forms nucleoprotein filaments on damaged single stranded DNA, protecting the DNA. RecA acquires protease activity, thus activating the self-cleavage of lexA protein

• This frees the operator and promoter site, thus facilitates gene expression of the SOS box gene.

Adaptive Repair

• It is an example of adaptive mutation in bacteria.

• Increased resistance to mutagenic and lethal effects of high doses of alkylating agents has been found in E. coli treated with sub-lethal concentrations of such agents for a long time.

• Adaptative mechanism of repair of mutation.

• DNA polymerase IV helps in adaptive repair.