DNA Repair- Lecture 30

Question 1

What are the types of damage?

Answer 1

mismatched base
modified base
covalent bonds between bases
double stranded break

Question 2

What is the source of mismatched base damage?

Answer 2

replication error

Question 3

What is the source of modified base damage?

Answer 3

spontaneous chemical

Question 4

What is the source of covalent bonds between bases damage?

Answer 4

UV light –> pyrimidine dimers

Question 5

What is the source of double stranded break damage?

Answer 5

XR, chemicals/bleomycin

Question 6

What repair systems are present for mismatched base damage?

Answer 6

3' endonuclease proofreading
mismatched repair (MMR)

Question 7

What repair systems are present for modified base damage?

Answer 7

base excision repair (BER)

direct repair

Question 8

What repair systems are present for covalent bonds between bases damage?

Answer 8

nucleotide excision repair (NER)

translesion synthesis

Question 9

What repair systems are present for double stranded break damage?

Answer 9

non-homologous end joining (NHEJ)

homology directed repair (HDR)

Question 10

Explain the activity of 3’ exonuclease in repairing for mismatched base pair damage.

Answer 10

DNA polymerase epsilon and delta has intrinsic exonuclease “proofreading” that cuts out the mismatched base that was just inserted and replaces the correct nucleotide before continuing

Question 11

Explain the mechanism of mismatch repair for mismatched base pair damage.

Answer 11

If the major DNA replicative enzymes don’t catch the error, MSH binds, recognizes the mistake and recruits a 3’ exonuclease to a nick nearby, DNA strand containing the mismatch is degraded, single strand gap is correct filling by DNA pol delta

Question 12

What is one example of a mutation in the mechanisms needed for repair for mismatched base pair damage.

Answer 12

HNPCC (Hereditary Non-polyposis Colon Cancer)

Question 13

Explain the mechanism of base excision repair for modified base damage.

Answer 13

glycosylases recognize unnatural bases in DNA, cut the bond between the base and the sugar (1’C) –> apyrimidinic site which can be recognized by an AP endonuclease –> makes a nick in the phosphate-sugar backbone allowing deoxyribosephosphodiesterase to remove the phosphate-sugar –> DNA polymerase beta fills in the correct nucleotide –> ligase closes up nick

Question 14

Explain the mechanism of direct repair for modified base damage.

Answer 14

methylated bases are recognized by direct reversal proteins which bind to methylated base and transfer the methyl group to a Cys residue in its active site

Question 15

What is a pyrimidine dimer?

Answer 15

consecutive pyrimidine rings are covalently linked to each other resulting in terrible kink in the DNA, blocking replication and transcription

Question 16

Describe the nuceotide excision repair (NER) system for covalent bonds between bases damage.

Answer 16

an XP (xeroderma pigmentosum) protein recognizes distorted DNA region –> other XP proteins unwind and excise a patch of DNA which is then filled in by DNA polymerase epsilon or delta

Question 17

What is an example of a mutation in the repair mechanisms for covalent bonds between bases damage?

Answer 17

xeroderma pigmentosum, which is caused by inherited mutation in the nucleotide excision repair genes and causes significantly increased risk of developing thyamine dimerization in skin cells leading to multiple basal cell carcinomas and other skin malignancies frequently at young ages
pts must avoid sun

Question 18

Describe translesion synthesis for repair of covalent bonds between bases damage.

Answer 18

DNA polymerases zeta and eta (which are error prone) are able to replicate past the pyrimidine dimer to either put in the correct base opposite the dimer (normally eta) or incorrect bases (normally zeta)- which still outweighs the disastrous effect of a block to replication

Question 19

Why does double-stranded breakage have more important consequences than any of the other kinds of damage?

Answer 19

because it is incompatible with the cell’s mechanism of equally distributing chromosomes to daughter cells
because repair is based on the integrity of the complementary strand

Question 20

Explain the mechanism for repair of double strand breakage by non-homologous end joining (NHEJ).

Answer 20

Ku proteins in complexes bound to two ends forms a synapse; helicase activity of Ku unwinds both ends; short single-stranded region in one DNA base pairs with homologous region in the other creating a microhomology region; unpaired 5’ regions are removed, then free ends are ligated
normally results in small inserts or deletions

Question 21

Explain the mechanism for repair of double stranded breakage by homologous recombination.

Answer 21

endonuclease chews at break to generate a single stranded DNA –> ssDNA is bound by replication protein A (RPA) with Rad51 protein –> BRCA2 catalyzes the replacement of RPA with Rad51 –> BRCA1 (in complex with BARDI) stimulates invasion by Rad51-coated ssDNA of homologous doouble stranded DNA (from sister chromatid) which is used as a template for repair

Question 22

What is an example of a mutation in the mechanism of repair for double stranded breakage damage?

Answer 22

mutations in the genes that code for BRCA1 or 2 proteins result in breast cancer because they can no longer repair by homologous recombination

Question 23

Explain how gene editing is similar to natural mechanisms utilized for repair of damage.

Answer 23

CRISPR-Cas9 works similarly to homologous recombination
single-guided RNA binds CRISPR-Cas9 complex and directs enzyme to a complementary DNA sequence –> Cas9 endonuclease cleaves the target DNA, leaving a double stranded break which can be repaired by homologous recombination with donor DNA provided as template strand OR by NHEJ (resulting in an indel)

Question 24

What is CRISPR-Cas9?

Answer 24

a gene editing tool that allows for one to target a specific double-stranded DNA break in a gene of interest and repair it