4.) DNA Repair

Question 0

What nucleotide base can be modified by one chemical reaction to form another nucelotide base?

Answer 0

Cytosine —> Uracil

Deamination reaction.

Question 1

What is de-amination?

Answer 1

This is the removal of an amine group from an organic molecule, usually to be replaced by a carbonyl/hydroxy group.

Question 2

What nucleotide is easily susceptible to epigenetic mutation?

Answer 2

5-methylcytosine, which is easily converted to thymine through deamination.

Question 3

What are three common causes for spontaneous alteration that will require DNA repair?

Answer 3

1. ) Spontaneous oxidative damage
2. ) Hydrolytic attack (depurination or deamination)
3. ) Uncontrolled methylation by S-andeosylmethionine

Question 4

Induction of UV light onto DNA often results in the formation of…

Answer 4

Pyrimidine dimers (thymine usually), and subsequently a bend/kink.

Question 5

What are the 4 repair mechanisms that we went over in this lecture?

Answer 5

1. ) Mismatch repair
2. ) Base excision repair
3. ) Nucleotide-excision repair
4. ) Homologous recombination

Question 6

True or False: The mutations that we are discussing in this lecture are mutations in DNA replication.

Answer 6

False. These are mutations that occur after replication has taken place.

Question 7

At what point would you find hemimethylated DNA?

Answer 7

Shortly after replication, the template strand is methylated whereas the daughter strand is not.

Question 8

What enzyme is responsible for methylation of DNA?

Answer 8

Dam methylase

Question 9

In methylation and mismatch repair what protein recognizes the GATC hemimethylated sequence? What protein detects the mismatched base pair?

Answer 9

MutH (req ATP)

MutS (req ATP)

Question 10

Describe the process of methylation and mismatch repair.

Answer 10

There is a short period of time after replication when the daughter strand is unmethylated. The MutS protein runs along the DNA to find any mismatched base pairs. At the site of mutation, the MutS protein associates and will now form a complex with MutL. The DNA is cinched from both sides through the MutS an MutL complex until the complex runs into a methylation (detecting the template strand). MutH protein will bind to either side of the MutL and MutS complex. The structure looks like a loop at this point.
MutH will then cleave at the 5’ site of the G base on the unmethylated strand at the most proximal methylated site. This knick in the DNA is the point of excision repair. Depending on the side of the knick, you will either have a 5’—>3’ exonuclease or a 3’—>5’ exonuclease that will remove a portion of the daughter strand up to the mismatched based pair. DNA polyIII and the SSB (single stranded binding protien) will resynthesize the cleaved portion of the daughter strand and include the correct base pair.

Question 11

What is the most stable and standard of the repair mechanisms?

Answer 11

Methylation and mismatch repair. (note jsut something he mentioned in class, probably won’t be on the test).

Question 12

When would nucleotide excision repair be used?

Answer 12

When a portion of DNA is damaged by UV radiation (or carcinogens from cigarette smoking), forming a dimer.

Question 13

What is depurination?

Answer 13

This is the complete removal of a nucleotide base from its sugar residue.

Question 14

Nucleotide deletions often result from what type of chemical modification?

Answer 14

Depurination

Question 15

Nucleotide substitution mutations often result from what type of chemical modification?

Answer 15

Deamination

Question 16

When would base excision repair be used?

Answer 16

When a single nucleotide base is damaged.

*Often occurs with cystine since it easily deaminates to uracil.

Question 17

Describe the process of base excision repair.

Answer 17

DNA glycosylase detects the damaged base and will cleave the nucleotide base from the sugar backbone. An AP endonuclease then cleaves the phosphodiester bond near the 5’ end. DNA polymerase I then starts synthesis at the free 3’ hydroxyl removing and replacing a portion of the damaged strand. Ligase then connects the knick that is formed after DNA poly I dissociates.

Question 18

Describe the process of nucleotide excision repair.

Answer 18

DNA lesion forms due to the mutation. Excinuclease will cleave a phosphodiester bond upstream and downstream from the lesion on the damaged strand (29 units long in humans, 13 in bacteria). DNA helicase then removes the damaged portion of DNA. DNA polymerase (I in E. Coli, epsilon in humans) will resynthesize that portion of the strand. Ligase will then fill in the knick.

Question 19

Non-homologous end-joining and homologous recombination are repair mechanisms that are used when…

Answer 19

There is an accidental double stranded break in DNA

Question 20

What is the most common form of repair for double stranded breaks? What is the problem with this type of repair mechanism?

Answer 20

Non-homologous end-joining. It results in a deletion of DNA.

Question 21

Describe the process of non-homologous end-joining.

Answer 21

Double stranded cleaved ends are recognized by Ku heterodimers. These recruit other proteins that will process the DNA to make blunt ends and then ligate them together.

Question 22

What is the only double stranded break repair mechanism that we use in somatic cells that aren’t dividing?

Answer 22

Non-homologous end-joining.

Question 23

When is the only time that homologous recombination repair of a double stranded break will occur?

Answer 23

S phase or early G2 phase. Need sister chromatids to act as templates

Question 24

Describe the process of homologous recombination repair of double stranded breaks.

Answer 24

An exonuclease discovers these breaks and will degrade the 5’ ends, leaving a 3’ over hang. The sister chromatid will then form a branch point with the damaged DNA so that it can be used as a template. The 3’ is elongated, the sister chromatid moves up the strand (branch migration), meanwhile the newly synthesized DNA will match with its complementary strand. This allows the top strand to now be elongated by a DNA polymerase. Ligase will seal the ends. Now have an accurate, newly synthesized DNA strand. No mutation/deletion.

Question 25

Much of the formation of cancer based on genetics is a result of…

Answer 25

a failure to repair a mutation.

Question 26

BRCA1 (breast cancer susceptibility 1) proteins are involved with…

Answer 26

binding and repairing double stranded breaks by invoking homologous repair. If these double stranded breaks are detected, the cell cycle will halt until BRCA1 or BRCA2 fix the break. If they can’t fix the break, the cell will induce apoptosis.

Question 27

How does a a mutation in BRCA1 or BRCA2 contribute to the formation of cancer?

Answer 27

A deficiency in BRCA1 will impair the DNA repair system, and will activate P53 to induce apoptosis. However, if P53 is mutated or inactivated, it will circumvent halting the cell cycle and apoptosis. This leads to clonal expansion and tumorigenesis.

Question 28

P53 proteins are involved in…

Answer 28

DNA repair, these proteins will halt the cell cycle so that DNA can be repaired before it is replicated or if the DNA can’t be repaired they will induce apoptosis. For instance they will respond when DNA is damaged by UV radiation.