lecture 24 - DNA damage & repair

what are mutations

alternations in DNA structure can produce permanent changes in genetic info encoded if not repaired

examples of agent causing mutation

• environment agents: UV light, reactive oxygen species, ionizing radiation

• chemical agents

two types of mutations

1. point mutations: substitution of one base pair for another, usually caused by modification/damage of bases

2. insertions/deletions: of one or more base (“indels”), often caused by “DNA intercalating” agents

three types of point mutation

• silent: still codes same amino acid

• missense: codes for a different amino acid, can be mild - severe

• nonsense: codes for a premature stop codon, cuts protein short and stop function, very severe

how do point mutations even happen ?

when template is damaged so its H-bond donor/acceptor arrangement favours a base other than the one it normally paris with

two types of base substitution:

• transition

  • most common

  • purine replaced w/ a purine(e.g. G or A) or pyrimidine replaced w/ another pyrimidine(e.g. C or T)

• tranversion

  • not common

  • purine replaced w/ pyrimidine or vice versa

2 chemical mutagens that modify bases and can lead to point mutations & how they do it

• deamination

• alkylating agents

modifications change H-bonding ability of the base, so they change DNA template leading to mutation

deamination

deaminating agents: nitrous acids

• deaminates aromatic primary amines

• Treatment of DNA with nitrous acid results in deamination of adenine, conversion to hypoxanthine. While adenine base pairs with thymine, hypoxanthine base pairs with cytosine because it can form two H-bonds with cytosine. Thus, deamination induces a mutation from A-T to G-C.

• deamination can also occur spontaneously

  • cytosine can become deaminated to form uracil, resulting in a C-G → T-A mutation after replication.

what method can repair deaminated bases

base excision repair

alkylating agents

• alkyalting agents add a methyl/ethyl group, usually to purines

• alkylating agents: S-adenosylmethionine, dimethylsuflate, dimethylnitrosamine

• normal cellular metabolism can produce reactive oxygen speices (superxoide ion O2-, hydroxide radical, OH) which can also cause DNA alkylation

• O6-alkylguanine is highly mutagenic bc it base pairs with T instead of C (GC→AT mutation after replication)

what method can repair deaminated bases and how

direct repair

• Usually repaired before mutation occurs

• by DNA repair enzyme O6-alkylguanine alkyltransferase, which transfers the methyl group to a cysteine in its own active site

whats the chemical mutagen that causes insertion/deletions

intercalation agents

intercalation agents

• ethiduim bromide (EthBr)

  • intercalating agent used to visualize DNA on agarose gels

  • intercalates between bases, becoming intensely fluorescent when exposed to UV light

• Flat aromatic molecules such as acridines can intercalate between adjacent base pairs in the DNA double helix. Their presence can lead to the insertion or deletion of one or more base pairs.

how does ultraviolet and ionizing radiation damage DNA

• ionizing radiation: UV light, x rays, gamma rays, cosmic rays

• cause ring opening and breaks in sugar phosphate backbone of DNA (strand breaks)

• UV light induces the condensation of 2 ethylene groups (C6=C5) in adjacent thymines on the same DNA strand to form a cyclobutane ring

  • adjacent thymins dimerize to form a cyclobutane thymine dimer which block replication and transcription because it causes helix distortion (kink) blocking polymerization machinery

how can UV induced thymine dimers be repaired in bacteria and plants

• in bacteria, this “lesion” can be corrected by a photo- reactivating enzyme photolyase that binds specifically to thymine dimers and uses visible light to hydrolyse the bonds linking the pyrimidine rings

• the ability of an organism to survive UV irradiation directly correlates with its ability to remove thymine dimers from its DNA

• photo-reactivation is a method of direct repair

how can UV induced thymine dimers be repaired in humans

• humans do not have photolyases

• thymine dimers are repaired by nucleotide excision and somtimes base excision repair

what can happen when thymine dimers are unrepaired

lead to melanomas (skin cancer)

ABC

• A - asymmetry

• B - border

• C - colour

consequence of defects in nucleotide excision repair enzymes

• people affected by a rare skin disease Xeroderma pigmentosum (XP) have mutations in a number of human nucleotide- excision repair genes (responsible for correcting thymine dimers)

• people with XP are extremely sensitive to sunlight or ultraviolet light - die from melanoma and other skin malignancies (cancers)

• changes in the skin become evident in infancy and worsen with time - skin becomes dry, and there is a marked atrophy of the dermis

explain oxidation damage to DNA by reactive oxygen species

• most prevalent soure of DNA damage

• reactive oxygen speices: hydrogen peroxide, hydroxyl radicals, & superoxide radicals

  • arise during irradiation(exposing an object, food, or person to energy waves or particles), or as a byproduct of aerobic metabolism

• can cause oxidization of deoxyribose and bases and strand breaks

how is damage from reactive oxygen species repaired

• DNA glycosylases using base excision repair

  • The human genome sustains tens of thousands of “oxidative hits” per day, most of which are repaired by ^

• cells have elaborate defense mechanism to convert reactive oxygen species to harmless products to prevent damage

• also DNA repair mechanisms to repair damage

5 mechanisms of DNA repair and brief description

1. Direct repair - damaged bases are not removed but repaired “on-site;” e.g., O6-alkylguanine alkyltransferase, E. coli photolyase for thymine dimers

2. Base excision repair - offending base is removed and replaced; e.g., bases modified spontaneously or by chemical mutagens

3. Nucleotide excision repair - offending nucleotides are removed and replaced; e.g., thymine dimers in eukaryotes

4. Mismatch repair – when the wrong nucleotide is added during DNA replication – system must discriminate between the template (correct) and the newly synthesized strand (incorrect); many enzymes involved

5. Homologous recombination (repair of double strand breaks, stalled replication,etc.)

direct repair

• fixes damaged genetic code without cutting the DNA backbone or needing a template

• examples:

  • Photoreactivation: Uses photolyase enzymes activated by visible light to break abnormal bonds in UV-induced pyrimidine dimers,

  • Alkylation Reversal: Uses O6-methylguanine methyltransferase to remove unwanted methyl or alkyl groups from guanine bases.

base excision repair

DNA glycosylase — role in base excision repair

→ Scans DNA and recognizes a specific type of damaged or incorrect base (e.g., uracil from cytosine deamination, or an oxidized base).

• Cleaves the glycosidic bond between the damaged base and the deoxyribose sugar, removing just the base

• Leaves behind an AP site (apurinic/apyrimidinic — a sugar with no base attached)

• This AP site is then recognized by downstream enzymes (AP endonuclease, polymerase, ligase) to complete the repair

• Different glycosylases exist for different types of damaged bases (specificity matters)

nucleotide excision repair

Enzymes that complete excision repair

Both base excision repair (BER) and nucleotide excision repair (NER) rely on the same general enzyme toolkit after the damage is recognized:

• Nucleases — cut out the damaged region (in BER: an AP endonuclease cuts near the AP site left by glycosylase; in NER: nucleases cut on both sides of the damaged nucleotides, removing a short stretch)

• DNA polymerase — fills the resulting gap by synthesizing new DNA using the intact complementary strand as a template

• DNA ligase — seals the final nick, forming the last phosphodiester bond to restore an intact strand

• Same "cut → fill → seal" logic as nick translation in replication, just triggered by damage instead of Okazaki fragment junctions

mismatch repair

Mismatch repair — how the system knows which strand is wrong

After replication, the mismatch repair system must correct only the newly synthesized (daughter) strand, not the original template — but both strands now have "normal" bases, just paired incorrectly, so how does it know which one to fix?

• The system uses transient strand markers to distinguish old vs. new DNA (e.g., in bacteria, the parental/template strand is methylated while the newly made daughter strand is not yet methylated)

• This temporary asymmetry lets mismatch repair enzymes specifically target and excise the nucleotide on the unmethylated (new) strand, then resynthesize it correctly using the methylated (template) strand as the guide

• Without this discrimination mechanism, the repair machinery could just as easily "fix" the correct base and lock in the mutation

homologous recombination

trimming the broken ends, using a matching homologous sequence (like a sister chromatid) as a template, and synthesizing new, exact DNA to bridge the gap