DNA Damage and Repair

Xeroderma Pigmentosum

a rare genetic disorder that causes extreme sensitivity to ultraviolet (UV) radiation, leading to a high risk of skin and eye cancers.

Mutation is

an accidental, permanent change in a DNA sequence that is rarely advantageous

nonsense mutation

single base substitution that introduces a stop codon

missense mutation 

single base substitutions that replace one amino acid with another

silent mutation

single base substitution that changes the DNA sequence without altering the encoded protein

Frameshift mutation

insertion or deletion that is not a multiple of three bases

What is DNA damage?

abnormal chemical structure of DNA

DNA damage can result in

mutations

_______ promote changes in DNA sequences

Mutagens

Ames test determines if a compound is a

mutagen

How does the Ames test work?

Strain of salmonella with inactive enzyme of histidine biosynthetic pathway. Needs histidine in growth media to grow. Cells that grow without histidine had a spontaneous mutation.

________ of C → U and 5 meC → T are most common DNA base damage and can occur spontaneously

Deamination

5-meC is commonly found in

CPG island (mutational hotspot)

Mutagens are often also…

carcinogens

Reactive oxygen species are generated by

cellular respiration

Most common ROS is hydroxide free radical inserts into

G or T (OXIDATION!)

Depurination is

the hydrolysis of the glycosidic bond linking a purine base to the sugar phosphate backbone

Depurination yields

an abasic site (site without a base) or AP site (apurinic/apyrimidinic site)

Alkylating agents _______ modify bases in DNA and distorts the heli

covalently

Sulfur mustard (mustard gas)

environmental alkylating agent

Spontaneous alkylation by ___________ of G → 7 methylguanine

S-adenosylmethionine

Does 7-methylguanine belong in DNA?

NO, it is located in the 5’ methyl cap in RNA

_________ _________ is most common DNA damage caused by ultraviolet radiation

Thymine dimers

What is ionizing radiation?

high energy radiation that can release electrons from atoms generating ions which can break covalent bonds

Ionizing radiation can cause what kind of breaks

ss, ds, and damaged bases at break sites that DNA ligase cannot process

Mismatch Repair (MMR) corrects

mismatched base pairs mainly from errors in replication

MutL-MutS complex recognizes

mismatch between base pairs

Muth identifies

hemimethylated parental GA TC strand and newly synthesized strand

In the removal of the mismatch, exonuclease

digests from nick through the mismatch

In the removal of the mismatch, DNA Pol III

fills the gap

In the removal of the mismatch, DNA ligase

seals the nick

Base excision repair (BER) repairs

mismatches resulting from damaged bases, depurination, and ss breaks in DNA

In BER, DNA glycosylase cleaves

the glycosidic bond making an abasic site (AP site)

In BER, AP endonuclease initiates

repair of abasic sites by making a ss break in DNA at the abasic site

In BER, DNA Pol…

has both 5’ → 3’ exonuclease and the DNA synthesis activities

In BER, DNA ligase

seals the nick

Nucleotide Excision Repair (NER) repairs

lesions that distort DNA double helix, such as thymine dimers or alkylation

In NER, excinuclease

hydrolyze two phosphodiester bonds, one on either side of the distortion

Excinuclease in prokaryotes

UvrABC excinuclease

Excinuclease in eukaryotes

XP excinuclease, large complex that contains XP-A through XP-G subunits plus TFIIH helicase

In NER, helicase

excises the damaged DNA leaving a gap

Helicase in prokaryotes for NER

UvrD helicase

Helicase in eukaryotes for NER

TFIIH

What is cockayne syndrome?

mutations in TFIIH subunits, like XPB and XPD, can cause the syndrome and is a rare and fatal autosomal recessive neurodegenerative disorder characterized by growth failure.

In NER, DNA polymerase I or DNA polymerase E

fills in the gap

In NER, DNA ligase

seals the nick

Non-homologous end joining (NHEJ) is the predominant mechanism in G0 and G1 for

repair of double strand breaks in mammalian cells

T/F: NHEJ is error prone repair

TRUE

In NHEJ, Ku70/80 complex binds

loose ends of DNA

In NHEJ, PKcs is a kinase that

initiates bridging of the broken ends

DNA pol micro and lambda add bases and are…

error prone terminal transferases

In NHEJ, DNA ligase

seals the nick

Homologous recombination repair (HRR) can repair

ds break in DNA after the DNA has been replicated

Template for HRR is

sister chromatid

BRCA1/2 in HHR is involved in

strand invasion

T/F: HHR is an error-prone repair mechanism

FALSE: error-free because no genetic information is lost