DNA Replication.

Question 1

In what direction does DNA replication happen in? What bonds for formed from when new DNA joins the original strand? What are parts of these new pieces of DNA called? And what do they bind to in particular?

Answer 1

This happens in a 5 prime to 3 prime direction. Remember 53

This forms phosphodiester bonds between the new DNA pieces and the original DNA

These new pieces are DNA have a region called deoxynucleotide triphosphates DNTP

These bind to the 3 prime end of the DNA strands. The hydroxyl bit.

(Note the DNTPs make up part of the new DNA molecule)

Question 2

What happens when the DNTps are used to bind to the template DNA strand?

Answer 2

The incoming DNTps undergo nucleophilic attack from the three prime hydroxyl

This allows for a phosphodiester bond to form

Question 3

What are the two original DNA strands called? In what direction do they run in reference to one another?

Answer 3

There is the primer strand and the template strand

These run in opposite directions to each other (as in 5 to 3 prime ends are in opposite directions)

Thus the strands are antiparallel

Question 4

What is the formation of new DNA strands dependent on?

Answer 4

They are dependent on a sliding clamp

DNA polymerase

A lagging and leading strand

DNA helicase and primase

DNA ligase

Question 5

Note about E coli DNA rep and human DNA rep?

Answer 5

They are highly conserved

Question 6

How does DNA replication begin? And when does it begin?

Answer 6

Note this is sooner Then you think

You have a DNA strand which is unwound by DNA helicase

This creates a replication fork between the two DNA strands

As each DNA strand is unwinding, this is when DNA replication is also occuring

(So note DNA rep doesnt happens when the strands have fully separated. It occurs as the strands are unseparating)

Question 7

What is important to note about DNA attachment to the uncoiling original DNA strands?

Answer 7

Both uncoiling strands dont have new DNA attached to them at the same time. These happen at different times.

Question 8

What is important to note about the DNA’s antiparallel arrangement?

In ref to the replication fork and 5 prime ends

Answer 8

One of The original DNA 5 prime ends is away from the replication fork and one of the ends is at the replication fork

Question 9

Describe the leading strand of DNA in DNA rep

Answer 9

The leading strands 5 prime end is away from the replication fork

This strand can be continuously synthesised without being interrupted

New pieces of DNA join as we move into the DNA replication fork

Question 10

Describe the DNA lagging strand? From where to where does synthesis occur?

Answer 10

Synthesis occurs from the replication fork outwards to the end of the DNA molecule

In the 5 to 3 prime direction

Question 11

Why is the lagging strand considered to be made in a discontinuous manner?

Remember DNA replication happens as soon as the DNA helicase has BEGUN uncoiling the original DNA strand into two pieces.

Answer 11

The lagging strand is synthesised out from the replication fork

This means it has to wait for more of the DNA to uncoil (in comparison to the leading strand) before it can be synthesised

The lagging strand begins to be synthesised when enough of the DNA template strand is revealed by the DNA helicase

but it then must pause to wait for more of the original DNA strand to uncoil and become exposed before more synthesis occurs.

Thus synthesis is discontinuous.

Question 12

What are okasaki fragments?

Answer 12

The lagging strand synthesises DNA in pieces

These are called okasaki fragments

These fragments must be joined together

Question 13

Summary of leading and lagging strand?

Answer 13

The leading DNA strand is synthesised in a continuous manner from the 5 to 3 prime

The lagging DNA strand is synthesised in a discontinuous manner in a 5 prime to a 3 prime direction from the replication fork

Question 14

What synthesises new DNA strands? And what does this enzyme bind to?

Answer 14

New DNA strands are synthesised by DNA polymerase

The DNA polymerase first binds to a RNA primer at the beginning of the DNA strand and work from there

The primers are bound to the original DNA strand.

Question 15

How do RNA primers form? How many primers are needed for the leading and lagging strand of DNA?

Answer 15

They form due to primase enzymes

These use template DNA and nucleotide triphosphates to make these primers

The leading strand of DNA only needs one RNA primer

The lagging strand needs a RNA primer for each okazaki fragment.

Question 16

What does DNA ligase do?

Answer 16

Seals the gap between okasaki fragments

This converts the okasaki fragments to a continuous strand

Question 17

How does DNA polymerase bind to the many primers on the lagging strand of DNA? And how does DNA ligase close the gap?

Answer 17

DNA polymerase binds to the RNA primer to synthesise part of the lagging strand. (One okasaki fragment)

After this fragment is synthesised , ribonuclease H removes the RNA primer

DNA polymerase then extends across the okasaki fragments gap (note it doesnt close the gap)

The gap is closed by DNA ligase.

Question 18

What does DNA helicase use to unwind the DNA strand? And what does it have attached to it?

Answer 18

The DNA helicase uses ATP

It has an attached ATPase to hydrolyse ATP

Question 19

What is an example disease caused by the mutation of a DNA helicase?

Answer 19

Werner syndrome
- this is premature aging
- these mutations are autosomal recessive mutations (affects both genes on a chromsome)
These occur in the RECQ helicase gene known as WRN

Bloom syndrome?
- this is a cancer sydnrome which is caused by a loss of function mutation in the RecQ family DNA
This maintains genome integrity
- patients are very sensitive to UV light and thus when DNA is repairing or replicating it undergoes damage.

Question 20

What does the sliding clamp do? How does it enhance processivity?

Answer 20

Makes the processivity of DNA polymerase better

Question 21

What is the difference between non processive and processive DNA polymerase?

Answer 21

Non processive polymerise 1 Nucleotide per second

Processive polymerise 1000 nucleotides per minute

Question 22

How does the sliding clamp work? Where does it bind? What does it do?

Answer 22

It attaches to the DNA polymerase. This is at a position close to the primer template junction by a clamp loader

The sliding clamp binds around the DNA strand.

This keeps the DNA polymerase clamped to the DNA strand.

The clamp encirlces the DNA like a nut on a bolt helping move the DNA polymerase forwards.

Question 23

What are single stranded DNA binding proteins (SSBs)? What is the exmaple in humans? What do they do?

Answer 23

These increase the processivity of DNA polymerase also

In humans one example is RPA - replication protein A

The proteins expose single strands of DNA in the replication fork

This allows for template synthesis.

Question 24

What are hairpins?

Answer 24

These are short regions of the DNA template which form due to proteins like the single stranded DNA bind porteins not working

These stop the DNA polymerase from carrying out its functions.

It thus affects PROCESSIVITY

Thus SSBs stop hairpins forming and jeep the replication fork open

Question 25

What does DNA topoisomerase do? What does this help? What causes DNA tangling?

Answer 25

This stops DNA from being tangled during replication

This helps DNA polymerase’s processivitiy remember

Tangling is caused by super helical tension at the replication fork.

DNA toposiomerase reduces this tension and seals the backbone of the parental helix.

Question 26

What does DNA topoisomerase type 1 and type 2 do?

Answer 26

Type one nicks and reseals one of the two DNA strands backbone. This stops DNA tangling
- ATP is NOT needed

Type two nickes and reseals two of the DNA strands. This stops tangling of the DNA
- ATP is needed

Both relieve tension by causing loops to be resealed

Question 27

What starts DNA replication? And how many origins of replication are there? How many replication forks are on a DNA strand?

Answer 27

The recruitment of replication initiator proteins to the DNA strand

There are 1 replication of origin in E coli
600-700 in yeast and 100k in humans

There are TWO replication forks on DNA. One either side moving inwards.

Question 28

What are replication origins? And what does it mean if they are dormant?

Answer 28

These are just places on the DNA where replication occurs. Where the RNA primers bind for DNA polymerase to bind to

You can have active or dormant replication origins

The dormant origins are there in times of DNA stress. To ensure DNA is replicated.

Question 29

Yeast replication origins? Human replication origins

Answer 29

Yeast are Referred to as ARCS - autonomously replicating sequences

Human origins are referred to as LMNb2 or MYC

Question 30

Initiation of eukaryotic DNA replication is Biphasic, what does this mean? What happens in G1 and S phase?

Answer 30

This means initiation of replication can be seen in G1 and S phase of the cell cycle.

Replicator selection occurs in G1 phase - this is when pre-replicative complex form

In S phase - origins of replication are made active - DNA unwinds and recruits DNA polymerase

Question 31

What does the biphasic nature of DNA replication initiation ensure?

Remember this just means initiation of replication can be seen in G1 and S phase of the cell cycle.

Answer 31

It ensures the each chromosome is replicated at-least once per cell cycle

Question 32

What does the origin recognition complex do? (ORC)

In the formation of the pre replicative complexes.

Answer 32

ORC binds to the replicator sequence in the G1 phase to form the pre replicative complexes

The ORC causes the Helicase Loading proteins Cdc6 and cdt1 to bind to the pre replicative compelx

This also makes helicase loading proteins like Mcm27 to bind to the pre replicative complex

Question 33

What do the cyclin dependent kinases do in S phase of the cell cycle? Also Pre - replicative complexes are said to fire, what does this mean?

In ref to DNA replication initiation?

Answer 33

They activate pre existing replication complexes but prevent new ones from forming.

This CDK in the S phase increases rapidly in S phase and phosphates many complexes here

Pre - replicative complexes are said to fire. This means that they become DNA replication origins thanks to the CDKS

Question 34

At the very end of DNA strand the RNA primer is lost? What does this causes

Answer 34

This causes a single stranded overhang (because there is no RNA primer at the last part of the DNA, the rest of the DNA strand can’t be bound to by DNA polymerase and thus synthesis stops)

DNA polymerase can’t extend to continue replication

Question 35

How do eukaryotic cells deal with the overhang caused by the lack of RNA primer at the very end of the DNA strand (meaning DNA polymerase can’t synthesise the full DNA stand)?

Answer 35

The enzyme telomerase adds a TTAGGG repeat to the end of the DNA sequence which compensates for the lack of RNA primer

This DNA polymerase can then polymerise the gap that hasnt yet been synthesised.

This stops the DNA strand from shortening.

Question 36

What are telomerases? And what they do?

Answer 36

These are a hybrid between RNA and proteins

They are a ribonucleotide protein

They act as a template for telomere repeat sequences to bind to. Called a telomerase shuffle.

Question 37

What is the telomere repeat sequence and what is the complement? And what can we notice from the complement?

Answer 37

Sequence is TTAGGG repeat is AAUCCCAAU

Note the complement has had 3 more nucleotides added. Meaning another telomere repeat has to be added

This happens for up to 200 nucleotides. Is telomere elongation stops valuable DNA from being lost.

Question 38

Again what does temporal separation, so initiation of DNA rep in G1 and S phase, allow?

Answer 38

Ensures DNA replication occurs exactly once per cell cycle.