R6 DNA replication

Question 1

What does each carbon in ribose interact with ?

Answer 1

1’ - base
2’ - OH in RNA, no OH in DNA
3’ and 5’ - phosphate

Question 2

What is a nick and a gap ?

Answer 2

A nick is one strand of the sugar-phosphate is broken, no missing nucleotides, the phosphodiester bond is broken between the nucleotides, needs to be sealed by ligase

A gap is a missing stretch of the nucleotide, usually occurs on the lagging strand. Needs to be filled in by DNA polymerase and then sealed by ligase

Question 3

Where do the arrows run in a diagram of replication ?

Answer 3

5’ to 3’

Question 4

What are the 3 types of ends on double stranded DNA ?

Answer 4

Blunt end, 3’ overhand, 5’ overhang

Question 5

What do nuclease, ligase and primase do ?

Answer 5

Nuclease – cleaves sugar-phosphate backbone
Ligase – joins sugar-phosphate backbone
Primase - synthesis of primers

Question 6

what type of junction does a replication fork show ? a repair intermediate ?

Answer 6

replication fork - 3 way
repair intermediate - 4 way

Question 7

What are the types of replicative helicases in prokaryotes and eukaryotes ?

Answer 7

DnaB is the replicative helicase (5’3’) in prokaryotes, MCM is the replicative helicase (3’5’) in eukaryotes.

Question 8

Why are helicases called molecular motors ?

Answer 8

They use ATP, they turn energy into movement

re-annealing afterwards does not require energy

Question 9

What form do free nucleotides come in ?

Answer 9

Triphosphate, so when it is de-phosphorylated it releases energy for replication

Question 10

What are the 3 types of replication ?

Answer 10

Conservative vs semi-conservative vs dispersive

Question 11

What does an exonuclease do ?

Answer 11

corrects the wrong base and puts the right one in

Question 12

What does topioisomerase do ?

Answer 12

works further upstream to prevent mechanical stress

Question 13

DNA synthesis can only happen in … to …

Answer 13

5’ , 3’

Question 14

What occurs on the lagging strand ?

Answer 14

• RNA primers are laid on the lagging strand, and synthesized by primase
• DNA pol I then removes the primers and fills the gap
• DNA ligase fills in the gaps

Question 15

What do single-strand binding proteins (SSB) do ?

Answer 15

Bing to ssDNA to prevent winding back

Question 16

What do sliding clamps do ?

Answer 16

hold the polymerase in place whilst nucleotides are being added

Question 17

What does DNA pol I do ?

Answer 17

removes RNA primers and replaces them with DNA

Question 18

What is the differences in the replication fork in eukaryotes to prokaryotes ?

Answer 18

leading and lagging strand is synthesized by different DNA polymerases

Okazaki fragments synthesis

the replicative helicase travels along the lagging strand in prokaryotes and the leading strand in eukaryotes

Question 19

What are telomeres ?

Answer 19

the ends of linear eukaryotic chromosomes. Highly repetitive G-C and prevent degradation

Question 20

What are shelterin proteins ?

Answer 20

protect the telomeres and cap the chromosomes as a whole

Question 21

What happens to telomeres with every replication cycle ?

Answer 21

the telomeres shorten due to the leading strand being shorter than the lagging due to the lack of ozahaki fragments

The shorter the telomeres the more limited cell proliferation is

The shortening of telomeres has a protective role against cancers, they limit proliferation of pre-cancer cells.

Cancer formation consists of stabilising the telomere length

Question 21

What can nuclease do ? What are the different types of nucleases ?

Answer 21

break the DNA sugar-phosphate backbone between the phosphate and the C3 or between the phosphate and the C5

There are ssDNA specific nucleases and dsDNA specific nucleases

Exonucleases begin cleaving at the ends (requires ends to the linear DNA) of the DNA (5’ and 3’ or 3’ and 5’), endonucleases break mid-strand (don’t require an ending)

Question 22

How can helicase and nuclease couple ?

Answer 22

Helicase can bind (as a substrate) with the nuclease, to degrade both or one strand. If one strand degrades then the 3’ overhang allows for a template for DNA repair.

Question 22

What do replicase, reverse transcriptase, transcriptasem RNA replicase do ?

Answer 22

Replicase – polymerases for DNA replication

Reverse transcriptase – telomerase and the ones coded by retrovirus

Transcriptase – RNA polymerases in prokaryotes and eukaryotes, primase

RNA replicase – RNA viruses other than retrovirus undergo RNA replication and code for RNA replicase

Question 22

What is the directionality of polymerase ?

Answer 22

All polymerase have 5’ 3’ directionality, so you cannot extend the 5’ end, you can only degrade it

Question 22

What is the proofreading activity of the DNA polymerase ?

Answer 22

-DNA polymerases has a proofreading activity, removes a wrong base, which is done by a 3’–> 5’ exonuclease

Proofreading activity can be removed, if mutation is needing to be high (to create mutants)

Processivity is the ability of the polymerase to extend a DNA strand before it dissociates from the DNA and needs to bind again, the affinity for DNA aka.

Question 22

What does nascent mean ?

Answer 22

made or derived from

Question 23

What is a clump ?

Answer 23

a closed ring which cannot fall off the DNA, it slides along the DNA

Most common is PCNA (proliferative cell nuclear antigen), can react with 3 proteins at a time, brings different enzymes to the replication forms, it is regulated by SUMOylation and ubiquitination

Question 24

How does clump loading occur ?

Answer 24

Clump loader binds to the DNA and then the clump, then the loader is released.

Clumps do not catalyse

Clump loader is an enzyme as it catalyses the opening and closing of the clump ring using ATP.