Section 3.1 DNA Damage and Repair

Mutation; Silent, nonsense, and missense

An accidental, permanent change in a DNA sequence; Rarely advantageous

Silent: Mutation only has an effect on the DNA level (due to wobble effect), not on protein

Nonsense: Mutation causes a premature stop in protein production

Missense mutation:

1. Conservative: Change in AA likely has little effect on protein because it has similar properties to original unmutated AA

2. Non-conservative: Change in AA likely has large effect on protein because it does not have similar properties to original unmuted AA

Insertion vs Deletion mutations

Can cause frameshift mutation: Insertion/deletion that is not a multiple of 3 bases; Often leads to an early stop codon; mutations that affect RNA splicing often generate frameshift

In-frame insertion/deletion: Addition/removal of multiple of three; typically doesn’t negatively affect protein since it’s just one AA

• Not always the case though; Ex, In-frame deletion of UUC (phe) codon in CFTR is most common cystic fibrosis mutation

DNA damage (WILL BE ON EXAM/Specifically the types of damages)

NOT the same as mutations; DNA damage can be fixed, not mutations

Abnormal chemical structure of DNA; Can be spontaneous, Often caused by environmental factors; Can result in mutations

Very common; our cells are exposed to 70k DNA damage events per day; less than 1 in 1k become mutations; Single-strand breaks and depurination is the most common type

Cancer is the result of an accumulation of mutations

Ames Test

Mutagens are agents which promote changes in DNA sequences; They are often also classified as carcinogens (Cancer causing agents)

Ames test determines if a compound is a mutagen; Uses Salmonella typhimurium with an inactive enzyme of the H biosynthetic pathway; Paper disks soaked in certain concentrations of the mutagen is placed in the middle of the plate; In H-absent growth media, cell that grow without the AA had a spontaneous mutation; Level of growth indicates strength of mutagen.

Interpreting the results: This test shows positive mutagen; Carcinogen testing requires live animals

1. Negative control still has growth; this is considered the base-level spontaneous background mutation rate

2. Low concentration showed large ring of growth because it was an optimal rate of DNA damage which allowed the cell to recover but not too many as to kill the cells

3. Middle - High concentration showed a ring of inhibition because DNA damage rate was toxic for the cell

Recall: DNA damage can naturally occur spontaneously in the cell, which is one of the reasons why we have a negative control plate

DNA Base Damage: Deamination

Deamination of C → U and 5-meC → T; Most common, spontaneous

Common chemicals like Sodium Nitrate/ite

Contained in preservatives, tobacco, cosmetic products, gastric juices, even veggies like spinach; Our repair systems are so good it doesn’t affect us

Recall: 5-meC can be found on CpGs (2.2), considered a mutation hotspot

DNA Base Damage: Reactive Oxygen Species (ROS)

Reactive Oxygen Species (ROS) generated as a result of cellular respiration

Most common ROS are hydroxide free radicals (·OH), which inserts into either G or T via oxidation reaction, which can result in DNA strand breaks

Damaged bases can cause Pols to add wrong bases

DNA Base Damage: Depurination

Depurination: Hydrolysis of the glycosidic bond linking a purine base to the sugar-phosphate backbone

Yields an abasic site (Site without base); Also called AP sites (apurinic/apyrimidinic site)

Much more common than depyrimidination

How DNA Damage Turns to Mutation (CpG example)

Ex. CpGs are considered mutation hotspots

Deamination DNA damage occurs → Repair system does not fix the damage → Replication occurs, one of the two replicants will contain the mutation

DNA Damage: Bulky Lesions & Crosslinking

Distorts DNA structures

Alkylating agents covalently modify bases in DNA; Ex, sulfur mustard (mustard gas); Alkylation distorts DNA double helix

Covalent linking means the DNA sequence is stuck and can’t be separated for replication/transcription

Spontaneous alkylation by S-adenosylmethionine (SAM) of G → 7-methylguanine (Unstable and can cause depurination)

Thymine dimers: Most common DNA damage caused by ultraviolet radiation; Results in formation of cyclobutane ring between two adjacent pyrimidine rings

Common for pairs of thymines forming a thymine-dimer; Cyclobutane ring kinks the axis of the DNA helix; Confuses Pol, will either pause or add a random base

Xeroderma Pigmentosum

Caused by mutation in XPA gene encoding protein for nucleotide excision repair (NER) which removes bulky DNA lesions like thymine dimers (UV rad).

Causes accumulation of structural mutations in DNA helix. Greatly increases risk of skin cancer.

DNA Damage: DNA Strand Break

Ionizing radiation: High-energy radiation that can release electrons from atoms generating ions which can break covalent bonds (Cosmic rays, X-rays, and radioactive materials)

Single-strand break (nick)

Double-strand break (May be staggered breaks)

Also causes damage to bases at break sites, meaning DNA ligase cannot process the breaks back together (All damaged bases must be removed before breaks can be reattached

General Pathway of DNA Repair

Most DNA repair mechanisms can be broken down into four distinct phases:

1. Recognition of the lesion

2. Excision of the lesion

3. Resynthesis of the DNA

4. Ligation of loose ends

Note: We’re taught the prokaryotic version (Aka prokaryotic proteins), but the Eukaryotic version follows the same general pathway (Just more complex when looked at in detail)

Mismatch Repair: Recognition and Removal of Mismatch

Recognition:

This pathway follows DNA pol during replication to double-check its work

Mismatch Repair (MMR) corrects mismatched base pairs mainly from errors in replication; Genetic defects in eukaryotic MMR are associated with Hereditary Non-Polyposis Colorectal Cancer

Nonfunctioning/mutated NMR pathways are ALWAYS associated with a diseased state

MutL-MutS complex recognizes the mismatch

MutH identifies parental GA^meTC strand and newly synthesized strand; MutH endonuclease nicks the unmethylated strand

Removal:

Nick can be 5’ or 3’ of the mismatch

Exonuclease digests from nick through mismatch

DNA Pol III fills the gap

DNA ligase seals the nick

Base Excision Repair: Removal and Resynthesis

Base excision repair (BER): Repairs mismatches due to damaged bases, depurination, and single-strand breaks in DNA

DNA glycosylase cleaves glycosidic bond making an abasic site (AP site); Or the site is already abasic due to depurination; Eukaryotes have many DNA glycosylases, including uracil-, 8-oxoguanine, and thymine-DNA glycosylases

AP endonuclease initiates repair of abasic sites by making a single strand break in DNA at the abasic site

DNA Pol I has both the 5’ → 3’ exonuclease and the DNA synthesis activities; DNA Pol I is a high-fidelity polymerase; In Eukaryotes DNA Pol β serves this purpose

DNA ligase seals the nick

Nucleotide Excision Repair

Primarily used to fix damage that distorts DNA structure; especially when involving more than one nitrogenous base

Proteins are named after the disease study (UV radiation and XP)

Nucleotide Excision Repair (NER) repairs lesions that distort the DNA double helix, such as thymine dimers or alkylation

Excinuclease: Hydrolyzes two phosphodiester bonds, one on either side of the distortion

• Prokaryotes: UvrABC excinuclease

• Eukaryotes: XP exonuclease is a large complex that contains XP-A through XP=G subunits plus TFIIH helicase

  • XP: Xeroderma pigmentosum

Helicase excises the damaged DNA leaving a gap

• Prokaryotes: UvrD helicase

• Eukaryotes: TFIIH (NER and Transcription is interconnected; Increases accuracy of transcription)

  • Cockayne syndrome (One of the proteins involved in interconnection is nonfunctional; associated with higher risk of developing mutations due to less surveillance of genome)

DNA Pol I (E. coli) or DNA polymerase ε (humans) fills the gap

DNA ligase seals the nick

Non-Homologous End Joining in Eukaryotes

A process used to repair double-strand breaks

Non-Homologous End Joining (NHEJ) is the predominant mechanism in G0 and G1 (1.2, S28) for the repair of double-strand breaks in mammalian cells; Error prone repair

Ku70/80 complex binds loose ends of DNA

PKcs is a kinase that initiates bridging of the broken ends

Other factors bind to and process the loose ends to produce an area of “micro-homology” (sticky ends): Exonuclease will remove bases (including damaged ones); DNA pol µ and λ add bases (Error prone terminal transferases); Artemis endonuclease removes any overhanging flaps

DNA ligase seals the nicks

Since only 1.5% of our genome encode for protein, the cell is willing to risk the error. If unlucky, NHEJ ends up destroying the protein or affecting transcription

Homologous Recombination in Eukaryotes

Homologous Recombination Repair (HRR) can also repair a double-strand break in DNA after DNA has been replicated

HRR only can be used during S and G2 phases because the Template for this reaction is a sister chromatid; BRCA1/2 involved in strand invasion; Mutation in BRCA1/2 is highly correlated to cancer development (Especially Breast and Uterine cancer)

Strand invasion: Detecting, grabbing and bringing broken chromosome to sister chromatid for repair

No genetic information is lost as a result of HRR, so this is an error-free repair mechanism