DNA Damage and Repair

Mutation

an accidental, permanent change in a DNA sequence

Frameshift mutation

nsertion or deletion that is not

a multiple of 3 bases, usually leads to early stop

mutations that affect RNA

splicing often generate

frameshifts

What does In-frame deletion of UUC cause

cystic fibrosis

What does a mutagen do

promote changes in

DNA sequences

• Often these chemicals are also

cancer-causing carcinogens

Ames test

determines if a compound is a mutagen

• Uses a bacteria with inactivate enzyme of

the histidine biosynthetic pathway

• requires histidine in the growth media in

order to grow.

• cells that grow without histidine had a

spontaneous mutation

Deamination of C > U and 5-meC > T

Remove nitrogen

Reactive oxygen species (ROS)

generated by cellular respiration, and hydroxide free radical (•OH) inserts into

either G or T can lead to oxidation

Depurination

hydrolysis of the glycosidic

bond linking a purine base to the sugar-

phosphate backbone

• Yields an abasic (AP) site = site without base

How do alkylating agents impact DNA

Covalently modify DNA bases

• Environmental like sulfur mustard (mustard gas)

• Spontaneous alkylation by S-adenosylmethionine of G >

7-methylguanine

• unstable and can result in depurination

• Alkylation distorts the DNA double helix

What’s the most common DNA damage cause by UV radiation

Thymine dimers

Xeroderma Pigmentosum

caused by mutation in gene that codes for protein involved in nucleotide excision repair (NER)

usually supposed to remove thymine dimers caused by UV radiation

and fix distortions in DNA helix

Ionizing radiation

high-energy

radiation that can release electrons

from atoms generating ions which

can break covalent bonds

can cause single stranded or doubel stranded breaks that DNA ligase cant fix

The four phases of DNA repair

1. Recognition of the lesion.

2. Excision of the lesion.

3. Resynthesis of the DNA.

4. Ligation of loose ends.

Mismatch Repair (MMR)

corrects mismatched

base pairs mainly from errors in replication

MutL-MutS complex function in mismatch repair

recognizes the mismatch

MutH function in mismatch repair

identifies the parental GAmeTC strand

and newly synthesized strand

• MutH endonuclease nicks the unmethylated

strand

Mismatch repair mechanism after MutH endonuclease nicks unmethylated strand

• Exonuclease digests from nick

through the mismatch

• DNA Pol III fills the gap

• DNA ligase seals the nick

Base excision repair (BER)

repairs

mismatches resulting from damaged bases,

depurination, and single-strand breaks in

DNA.

DNA glycosylase

leaves the glycosidic

bond making an abasic site (AP site)

AP endonuclease

initiates repair of abasic

sites by making a single strand break in DNA

at the abasic site

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

What is the excinuclease in eukaryotes

XP excinuclease is a large complex that contains

XP-A through XP-G subunits plus TFIIH helicase

mutated in Xeroderma pigmentosum

What is the helicase in Eukaryotes

TFIIH

• Cockayne syndrome

Non-Homologous End Joining (NHEJ)

predominant

mechanism in G0 and G1 for repair of double-strand breaks

in mammalian cells

• error prone repair

Mechanism of Non homologous end joining

• 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

• DNA pol μ and λ add bases (error prone terminal transferases)

• Artemis endonuclease removes any overhanging flaps

• DNA ligase seals the nicks

Homologous recombination repair (HRR)

repair a double-strand break in

DNA after DNA has been replicated

What does HHR need for repair

• Template for reaction is sister chromatid

• BRCA1/2 involved in strand invasion