Genome Integrity 1

Question 1

What are 6 ways DNA can be mutated or damaged?

Answer 1

Tautomerization, Intercalating Agents, UV Light, Depurination, Hydrolytic Deamination, Alkylation, Oxidative Damage

Question 2

What kind of mutation occurs with tautomerization?

Answer 2

Adenine tautomerizes to bind C.
A - T
G - C

Question 3

What kind of mutation occurs with intercalating agents?

Answer 3

Base pair insertions and deletions since it unwinds DNA

Question 4

What kind of damage occurs with UV light?

Answer 4

Pyrimidine dimerization, causes bulky groups to form

Question 5

What kind of damage occurs from depurination?

Answer 5

Lose guanine and get an abasic nucleotide

Question 6

What kind of mutation occurs from hydrolytic deamination?

Answer 6

Cytosine -> Uracil from losing amino group.
C - G
T - A

Question 7

What kind of mutation occurs from alkylation?

Answer 7

Guanine gets methylated
O6-mG - T
A-T

Question 8

What are the 5 proteins involved in the Mismatch Repair System?

Answer 8

MutS, MutL, MutH, UvrD, Exonuclease

Question 9

Function: MutS

Answer 9

recognize mismatched DNA b/c mismatch is floppy rod

Question 10

Function: MutH

Answer 10

Binds hemimethylated DNA to recognize parent strand; nicks the non-methylated DNA

Question 11

Function: MutL

Answer 11

Two lobed protein that facilitates the connection between MutH and MustS

Question 12

Function: UvrD

Answer 12

Helicase that unwinds DNA so that the 5’ to 3’ exonuclease can chew up the DNA

Question 13

Mechanism: Mismatch Repair

Answer 13

1) Mismatch causes DNA to be bent and floppy, MutS binds mismatched DNA
2) MutL binds next to MutS
3) MutH binds hemimethylated DNA
4) MutL second lobe interacts with MutH, forming MutSLH complex; now active MutH can cut the non-methylated DNA strand
5) UvrD is recruit and unzips the DNA
6) Exonuclease comes in and cleaves DNA
7) Close gap by synthesizing DNA

Question 14

What do mutations in mismatch repair enzymes cause in humans?

Answer 14

Nonpolyposis colorectal cancer. Causes hereditary forms of cancer

Question 15

What kind of DNA damage does oxidative damage cause?

Answer 15

Strand breakage

Question 16

What are two examples of repair by direct reversal?

Answer 16

Photolyase and Dealkylation

Question 17

Mechanism: Photolyase

Answer 17

Proteins use sun energy to cleave UV-induced dimers to monomeric state. Found in every organism except placental mammals.

Question 18

Mechanism: Dealkylation

Answer 18

1) Ada protein transfers methyl from DNA to cysteine residue
2) Methylated Ada protein activates expression of its own gene
Feedback loop

Question 19

What are the 4 repair strategies for damaged DNA?

Answer 19

Base Excision Repair, Nucleotide Excision Repair, Translesion Synthesis, Recombination

Question 20

4 Proteins involved in Base Excision Repair

Answer 20

Glycosylase, AP Endonuclease, PARP

Question 21

Mechanism: Base Excision Repair

Answer 21

1) Damaged base is recognized by DNA glycosylase
2) Glycosylase cleaves the damaged base
3) AP endonuclease recognizes abasic site and cleaves phosphodiester bonds on both sides
4) Protein (PARP) recognizes nick and facilitates repair by DNA Pol

Question 22

What does Base Excision Repair Fix?

Answer 22

Single damaged bases

Question 23

4 Proteins involved in Nucleotide Excision Repair?

Answer 23

UvrA, UvrB, UvrC, UvrD

Question 24

Mechanism: Nucleotide Excision Repair (Bacteria)

Answer 24

1) UvrAB in complex binds the DNA lesion (or damaged site)
2) UvrB helicase activity unwinds DNA and UvrA is released
3) UvrC is recruited and cuts at 3’ and 5’ of damaged site
4) UvrD is recruited and helicase activity removes damaged DNA, UvrC is released
5) DNA pol comes in and synthesizes strand

Question 25

What does mutations in the proteins involved in Nucleotide Excision Repair cause?

Answer 25

Different mutations cause a large array of different phenotypes such as light photosensitivity and susceptibility to cancer

Question 26

How does translesion DNA synthesis work?

Answer 26

Large family of polymerases (Y family) that are highly error-prone allow replication to continue

Question 27

Why are translesions DNA polymerases so mutagenic?

Answer 27

Have a contact site that is very loose, allows the polymerase to get around the problem; this loose connection lowers the steric sensitivity of the polymerase lowering specificity of which base is added

Question 28

What are the 2 mechanisms of double strand break repair?

Answer 28

Non-homologous End Joining and Homology-Directed Repair

Question 29

When are the differences between the two mechanisms of double strand break repair?

Answer 29

1) NHEJ is preferred in G1, HDR is preferred after S phase

2) NHEJ is a mutagenic process due to imprecise joining.

Question 30

What are the 5 proteins involved in Non-homology mediated end joining?

Answer 30

Ku70/Ku80, DNA-PKcs, Artemis, Ligase IV, XRCC4, XLF/Cerannes

Question 31

Mechanism: Non-homology Mediated End Joining

Answer 31

1) Ends of ds break is slightly resected by nuclease to create ssDNA
2) Ku70/Ku80 (Ku heterodimers) binds broken ends and recruits DNA-PKcs
3) DNA-PKcs recruits Artemis and phosphorylates the protein
4) Phosphorylated Artemis trims single stranded tails
5) Ligase IV in complex with XRCC4/Cerunnos ligates the two ends, resulting in deletion

Question 32

How does homology directed repair work?

Answer 32

Homologous duplex strand invades and forms a D Loop. Get extension of strand that invaded until newly synthesized strand can anneal to strand that was displaced. Get DNA replication to fill in the gap.

Question 33

Which 4 proteins are involved in initiation of homology-directed repair and strand invasion?

Answer 33

RecBCD, Rec A

Question 34

Mechanism: Homology-Directed Repair (in E. coli)

Answer 34

1) RecBCD binds to ds break
2) RecBCD has helicase activity that will unwind DNA
3) While DNA is being unwound, RecB has nuclease activity that will degrade DNA
4) Once RecBCD hits chi sequence, 5’ tail is degraded to leave 3’ overhang
5) RecA-ATP is loaded onto 3’ tail to form presynaptic complex and RecBCD falls off
6) RecA-ssDNA nucleofilament binds to dsDNA and extends duplex to melt it
7) ssDNA displaces one stand to form D-loop
8) Elongation of ssDNA continues until replicating strand can anneal back to displaced strand

Question 35

Which enzymes resolve Holliday junctions?

Answer 35

RuvC in E. coli, GEN1 in eukaryotes

Question 36

Which enzymes move Holliday junctions?

Answer 36

RuvAB

Question 37

What is the ssDNA binding protein that facilitates strand invasion and D-loop formation in HDR? (3)

Answer 37

RecA (In E. Coli); Rad51 (eukaryotes); Dmc1 (meiosis)

Question 38

What kind of chromosomal rearrangements are produced by homologous recombination?

Answer 38

Intra-chromosomal deletion, unequal crossover between sister chromatids, unequal crossover between homologs, translocation between non-homologous chromosomes.