DNA Repair and recombination

Question 1

How often do mutations occur in human genomes during replicaiton?

Answer 1

roughly 1 nucleotide per 10^8

Question 2

What processes are able to correct errors in DNA replication?

Answer 2

• proofreading mechanism

- post replication repair

Question 3

Low fidelity and high mutation rates occurring within germ cells results in what?

Answer 3

low fidelity and high mutation rates in germ cells causes cell death

Question 4

Low fidelity and high mutation rates in somatic cells results in what?

Answer 4

Both will result in the formation and progression of cancerous cells

Question 5

In order for DNA polymerase to function properly what materials does it require?

Answer 5

• dATP, dGTP, dCTP, dTTP
• strands separated by DNA helicase
• primers on 3’OH ends

Question 6

What is the clamp loader protein?

Answer 6

• this protein hydrolyzes ATP in order to attach the sliding clamp onto the primer-template junction

Question 7

Explain how DNA mismatch repair occurs in humans.

Answer 7

Lagging Strand:

• sequence is found via broken segments,
• MutS finds the mutation, MutL finds the closest “nick” and the DNA section between the two is removed and repaired.

Leading Strand:
- unknown process

Question 8

DNA mismatch repair in E.Coli is flagged by what process?

Answer 8

The parental strand is methylated and signals that the other strand is the one that needs to be replaced.

Question 9

What is the process that DNA Topoisomerase Type 1 operates?

Answer 9

• covalently bonds to one strand, cuts the other strand and allows the DNA to uncoil about the bound phosphodiester bond. Reduces coiled tension

Question 10

How does topoisomerase 2 operate to relieve coiled tension?

Answer 10

• Used when two double helix cross paths.
• -It cuts and bind both strands of one helix to itself, undergoes a conformational change to pass the other helix between the cut, and then rejoins the helix that was doubly cut.

Question 11

Why is the repilcation origin often at sites with AT binding?

Answer 11

• AT only bond with two bonds, and is easily broken compared to GC.

Question 12

What consists of tightly packed DNA that is most often replicated late?

Answer 12

• heterochromatin, the tight packing makes the replication process proceed slowly

Question 13

Which DNA is loosely packed and is replicated faster and first?

Answer 13

-euchromatin is loosely packed

Question 14

What are the minimum requirements for an active Origin of Replication?

Answer 14

1. binding site for Origin recognition complex
2. AT rich zone
3. Binding site for origin recogniction complex attraction protein.

Question 15

What is a method to alter epigenetic inheritance?

Answer 15

-the recycling of histones from parental units, can retain some of the original patterning units and therefore pass on genetic material

Question 16

What is the purpose of telomerase?

Answer 16

The telomerase is an enzyme that adds a specific sequence to the end of the DNA sequence so the lagging strand is fully synthesized. It places a repeating sequence on the end that allows the binding of a primer and DNA polymerase.

Question 17

What are two syndromes that can result inf cancer due to defects in DNA repair mechanisms?

Answer 17

BRCA1: breast and ovarian cancer
BRCA2: breast, ovarian, prostate cancer

Question 18

What is Werner syndrome and what causes it?

Answer 18

• premature aging, cancer, and gene instability

- caused by mutation in DNA Helicase repair

Question 19

What is Bloom Syndrome?

Answer 19

• characterized by stunted growth, cancer, and gene instability
• caused by mutation in DNA helicase used in recombination

Question 20

What are the most common types of spontaneous DNA damage?

Answer 20

1. depurination: removal of base from nucleotide
2. deamination: removal of N from the base
3. dimerization: UV radiation causes pyrimidine dimer

Question 21

What types of repair processes can the cell use to fix damaged DNA?

Answer 21

1. base excision repair
2. nucleotide excision repair
3. transcription coupled repair

Question 22

Explain how base excision repair works, and when it is most commonly used.

Answer 22

1.used with depurination mutations with the AP endonuclease.

2. DNA glycosylase recognizes and removes the glycosyl bond
   - endonuclease and phosphodiesterase finish cleaving the rest of the strand to remove the damage.
   - AP endonuclease replaces damage with proper sequence

Question 23

How does nucleotide excision repair work?

Answer 23

1. an entire section of DNA strand is remoed, and is best for pyrimidine dimers, or chemically induced mutations.
2. DNA helicase, polymerase, and ligase are required in order to cut, unwind, and repair the damaged section.

Question 24

Why is transcription-coupled repair useful to a cell?

Answer 24

• repair machinery is paired with transcription equipment. This helps RNA polymerase, if it encounters damage to the DNA it is reading. The repair machinery will repair the lesion, probably with mutation, but transcription will continue

Question 25

Why in the humans is it acceptable to use transcription-coupled repair if there is a high likelihood it introduces mutation?

Answer 25

-only 1.5% of the genome is expressed as extrons, the remainder can be mutated to some extent and is better to have those mutations than it is to not have transcription taking place.

Question 26

What is Cockayne's Syndrome?

Answer 26

- growth retardation, skeletal abnormalities, and UV sensitivity. - caused by defective transcription coupled RNA polymerase

Question 27

Why is RNA most likely to not be the genetic material?

Answer 27

-cysteine can undergo deamination causing it to appear as a uracil. If RNA were used there would be no way to distinguish between a mutated base pair of Uracil or a normal non-mutation

Question 28

Deamination of a methylated C produces what?

Answer 28

thymine, which will create a lesion as G will be paired with T

Question 29

Translesion Polymerase?

Answer 29

this polymerase functions just as DNA polymerase, however does not have a proofreading feature. Therefore it can transcribe lesions/mutation to DNA and allow transcription to continue with DNA Polymerase

Question 30

How can double strand breaks be repaired?

Answer 30

1. non-homologous end joining | 2. homologous recombination

Question 31

What is non-homologous end joining?

Answer 31

1. major repair in Humans, and broken ends are ligated with DNA ligase

Question 32

What are the processes that homologous recombination can be used to fix?

Answer 32

1. repair double stranded break 2. exchange and form new genetic sequences 3. mechanically ensure chromosome segregation

Question 33

How does homologous recombination repair a double stranded break?

Answer 33

1. exonuclease degrade the 5' end of both strands 2. the 3' end invades the other newly formed DNA strand, matches and elongates the damaged strand to fill in the gaps. 3. the 'damaged' strand now becomes the template to fix the other s-strand sequence. 4. once done the new strands are ligated together. this provides a very accurate way to repair

Question 34

How can homologous recombination contribute to DNA hybridization?

Answer 34

1. Rec A/Rad51 bind with ssDNA and helps find a complementary sequence strand on the duplex DNA 2. ssDNA invades and inserts itself into the duplex DNA forming a heteroduplex DNA 3. the complementary sequence must be 15 bp long in order to attach.

Question 35

Homologous recombination can be used to help polymerase continue when it runs into a broken section on the parental DNA strand. How?

Answer 35

1. 5' exonuclease trims back the sequence to prepare it for strand invasion 2. the overhanging 3' end invades the ds, synthesizes the required material, and detaches 3. the leading strand is acting somewhat like a lagging strand 4. the DNA polyermase is attached and transcription continues

Question 36

What are the 4 general features of non-homologous end joining?

Answer 36

1. no template required 2. repairs site mutations 3. can create translocations 4. preferred method in human DNA

Question 37

What are unique features of homologous recombination?

Answer 37

1. uses daughter DNA duplex as a template 2. no mutations/alterations occur at repair sites 3. can repair other DNA types of damage

Question 38

What is a holliday junction?

Answer 38

DNA intermediate during crossing over that contains 4 strands of DNA from 2 separate helices, which is quickly cleaved via RuvC in a process called resolution

Question 39

What is resolution?

Answer 39

DNA strand cleavage by endonuclease RuvC, during crossing over events or gene conversion

Question 40

Which event, crossing over or gene conversion, is most commonly seen in humans?

Answer 40

-gene conversion at 90%

Question 41

Both DNA strand of a Holliday junction are cut in the same direction. What is the final result?

Answer 41

1. gene conversion with minimal amounts of gene exchange

Question 42

If both DNA strands are cut in opposite directions, what is the final result?

Answer 42

1. the upstream portion and the downstream portions are able to swap, resulting in crossing over.

Question 43

When does gene conversion most likely occur and what is the primary outcome of this process?

Answer 43

1. occurs during heteroduplex mismatch repair. | 2. causes loss of one allele and duplication of others.

Question 44

What type of transposons are predominate in bacteria>?

Answer 44

DNA-only. allows sections of of DNA sequence to move to new sections. - ability to spread AB resistance. - the insertion site is duplicated, so if the sequence is excised, the genome will retain the original insertion site

Question 45

In DNA-only transposons 3 different scenarios can take place. What is the cause of each of these scenarios? 1. restore transposon 2. unrestored transposon 3. mutation inserted

Answer 45

1. transposons are restored with ds break repair 2. transposons are unrestored with homologous recombination 3. mutations are inserted with nonhomologous end joining

Question 46

What is unique about retrovirus-like transposons?

Answer 46

they function under the same premise as retroviruses but lack a capsid and are therefore unable to migrate to new cells.

Question 47

Review how nonretroviral transposons, conservative site specific recombination, and site-specific recombination occur

Answer 47

Lecture 5 DNA processes 3