Week 4: DNA Replication Part II

Question 1

What are the roles of initator proteins for replication in e.coli

Answer 1

1. Binds to origin
2. Helps helicase bind
3. Requires ATP

Question 2

What unwinds DNA?

Answer 2

Helicase

Question 3

What direction does the predominant DNA helicase move in?

Answer 3

5’-3’ along the lagging strand template

Question 4

True or False: Helicase is on the lagging strand not the double helix

Answer 4

TRUE

Question 5

What are SSB proteins and what does it do?

Answer 5

SSB proteinss are single strand binding proteins which keep DNA strands separated by preventing strands from H-bonding

Question 6

What is the importance of SSB proteins?

Answer 6

SSB proteins keep DNA strands separated since they have the ability to stick back together after separation by helicase

Question 7

Before DNA replication begins, what is required? Why is it needed?

Answer 7

An RNA primer made by primase. The primer is needed because DNA polymerase cannot start building from nothing.

Question 8

What is RNA primer made of?

Answer 8

A short sequence of nucleotides with free 3’OH end that will be used by DNA polymerase

Question 9

What direction does RNA primase read the the template strand in and what direction does it create the RNA primer in?

Answer 9

RNA primase reads the template strand in the 3’-5’ direction but makes RNA primer in the 5’-3’ direction

Question 10

True or False: RNA primer does not get removed later on in DNA replication

Answer 10

False. It is later replaced because RNA primer is made of RNA.

Question 11

What are the overall steps in bacterial DNA replication?

Answer 11

1. Origin of replication
2. Binding of initiator proteins
3. Unwinding by helicase
4. Binding of single-strand binding proteins
5. RNA primers made by primase
6. DNA polymerase
7. Sliding clamp holds polymerase onto DNA
8. Nick sealing by DNA ligase

Question 12

Helicase + primase = ?

Answer 12

primosome

Question 13

What direction does the new DNA grow in (i.e. what direction does DNA polymerase add nucleoside triphosphates?)

Answer 13

DNA adds nucleoside triphosphates in the 5’-3’ direction

Question 14

How are nucleoside triphosphates added onto the growing DNA strand?

Answer 14

Two phosphates (pyrophosphate) are removed and the remaining phosphate bonds to the OH on the 3’ end of the growing strand and the base pair matches up

Question 14

What are sliding clamps?

Answer 14

Sliding clamps helps to hold DNA polymerase in place as it synthesizes a new DNA strand (it tells DNA pol. to get its shit together)

Question 14

What are okazaki fragments?

Answer 14

Okazki fragments are incomplete strands of DNA that are synthesized on the lagging strand

Question 14

How are the Okazaki fragments on the lagging strand liked together?

Answer 14

The space (nicks) left by the removal of RNA primer is sealed by DNA ligase, from the 3’ end of one fragment to the 5’ end of another

Question 14

What is used to make new DNA strands?

Answer 14

dATP (NOT ATP)

Question 15

True or False: DNA helicase and primase always work together side by side

Answer 15

False. They work in conjuction but not always together.

Question 16

What is the whole machine of bacterial DNA replication called?

Answer 16

the replisome

Question 17

True or False: Directionality never changes

Answer 17

True.

Question 18

What tends to happen as helicase uwinds DNA?

Answer 18

As DNA is being unwound by DNA helicase, it begins to want to spin, but it cannot, so supercoiling & torsional strain increases

Question 19

What solves the supercoiling problem when DNA helicase unwinds DNA?

Answer 19

DNA topoisomerase cuts the DNA, lets it spin, then binds it back together. This is done in order to relieve tension

Question 20

Although primase creates RNA primers to begin DNA replication, what is is not good at?

Answer 20

Creating primers at the ends of chromosomes

Question 21

At the end of DNA replication, why is there an issue at the end of the lagging strand?

Answer 21

When the RNA primer is removed from the very last okazaki fragment, there is no way to add onto it (it's a 5' end) so the lagging strand ends up **shorter** than the leading strand.

Question 22

What solves the issue presented by the lagging strand at the end of DNA replication? How does it solve the problem?

Answer 22

**Telomerase** solves the problem by adding a repetitive sequence (**RNA template**) to the lagging strand *template* allowing for more space to continue DNA replication, enough to make up for the loss of sequence information caused by the shorter lagging strand, and causes G-rich ends

Question 23

What does telomere replication resemble?

Answer 23

**Reverse** **transcriptase**, because instead of using DNA to create RNA, you're using RNA to create DNA

Question 24

Telomerase abundant in **stem** and **germ-line cells** but not in _________ ______

Answer 24

Somatic cells

Question 25

What is the issue with telomeres in cancer cells?

Answer 25

Most cancer cells produce **high** levels of telomerase

Question 26

Why is it an issue for there to be a mistake in DNA replication (i.e. a strand with a wrong base pair)?

Answer 26

In future replications there will be a permanent mutation

Question 27

What are the rates of error in RNA polymerase and DNA polymerase? What does this mean for the human genome?

Answer 27

RNA polymerases have an error rate of appox. 1 in 10^4, DNA polymerases have an error rate of approx. 1 in 10^9. This means that the human genome is only changed about **3** nucleotides every time a cell divides

Question 28

What are the two mechanisms for DNA proofreading & repair?

Answer 28

1. 3'-5' **exonuclease** ("backspace") 2. **Strand-directed** mismatch repair

Question 29

How does DNA polymerase proofread the newly synthesized DNA strand it creates?

Answer 29

It scans the strand as it makes it and removes the misincorperated nucleotide. This is the **3'-5' exonuclease**. It has two active sites, one for **polymerizing** and one for **editing**.

Question 30

How does strand-directed mismatch work in DNA repair in eukaryotes?

Answer 30

If proofreading fails this is what is typically relied on. * MutS protein recognizes and locks onto DNA mismatch * proteins recuits MutL and scans DNA * sliding clamp aids in positioning structure onto the new strand via identification of a **nick** * MutL nuclease is then activated and removes the part of the strand that contains the error

Question 31

**True or False:** DNA can't get damaged after synthesis

Answer 31

False.

Question 32

Which human diseases can be linked to defects in repair mechanisms? A. Breast B. Colon C. Skin D. All of the above

Answer 32

D.

Question 33

What are some ways DNA can be damaged through **stressors**?

Answer 33

1. oxidation 2. radiation 3. heat 4. chemicals

Question 34

What is a pyrimidine dimer?

Answer 34

When pyrimidines (i.e. thymine) that are stacked on top of each other form a bond (**you do not want this!!**)

Question 35

What are some ways DNA can be damaged **spontaneously**?

Answer 35

1. **Depurination**: the removal of a purine as a result of water 2. **Deamination**: specific to **cytosine**, the removal of an amine group due to water resulting in uracil (DNA will have RNA in it because of this) -- however this is common!

Question 36

What can be caused by spontaneous damage to DNA?

Answer 36

**Deamination** changes C to U which causes switched base pair mutations in DNA replication. **Depurination** removes a purine which results in a missing nucleotide when DNA replicates.

Question 37

Name the two general mechanisms of DNA repair (not proofreadings, so *after* synthesis)

Answer 37

1. Base excision repair (BER) 2. Nucleotide excision repair (NER)

Question 38

Differentiate BER from NER

Answer 38

Base exicion repair replaces **one nucleotide**, nucleotide excision repair cuts out a **whole section** of the strand with the error and with the help of DNA polymerase and DNA ligase replaces the cut section and seals it together.

Question 39

What are the two ways of DNA repair for double-stranded breaks?

Answer 39

1. **Nonhomologous end joining (NHEJ)**: when both strands break DNA nuclease processes it which causes some lost info/nucleotides, but then both ends are joined by DNA ligase ("quick and dirty" type of solution) 2. **Homologous recombination HR**: when both strands break but there are undamaged homologous DNA strands available, the double strand break will be accurately fixed by using the undamaged DNA strands as a template (slower, but more accurate and best case solution)