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Molecular Genetics > Lecture 14 - DNA Replication and DNA Damage > Flashcards

Lecture 14 - DNA Replication and DNA Damage Flashcards

1
Q

DNA is semi conservative - what is this?

A

Seperation of duplex DNA strands and the formation of a new strand by DNA polymerase, using the old strand as a template

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2
Q

What is elongation performed by?

A

DNA polymerase 3

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3
Q

what are the 3 steps of replication?

A

initiation
Elongation
Termination

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4
Q

In e.coli what is the initiation site called?

A

oriC

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5
Q

Does e.coli replicate bidirectionally?

A

yes

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6
Q

In elongation what is the gamma complex?

A

unloads polymerase onto DNA template via clamp

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7
Q

In elongation what does the beta subunit do?

A

sliding clamp

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8
Q

what does the tal subunit do in elongation?

A

makes a dimeric polymerase

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9
Q

Is helicase on the replication fork?

A

yes

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10
Q

What does the “thumb” of the polymerase do?

A

Puts the DNA closer to the area where nucleotides can be added.

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11
Q

What does poymerase 3 reguire to begin synthesis?

A

A primer

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12
Q

What is the polarity of DNA polymerase?

A

5 - 3

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13
Q

what is the primer made from?

A

RNA

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14
Q

What does the sliding clamp do?

A

keeps it from falling of the DNA

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15
Q

How does elongation of the replication fork take place?

A

the polymerase binds and extends the leading strand 5-3’

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16
Q

How does the lagging strand get synthesised?

A

The lagging strand is synthesised 3 -5 prime but as its complementary to the smoothly synthesing leading strand then you get a non-continous product.

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17
Q

What are the non continous product in the leading strand called?

A

these are called ozaki strands

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18
Q

What happens to make the lagging strand continous?

A

A loop so the two polymerases can connect and move in the same direction.

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19
Q

How do you finish replication?

A

RNA primers must be removed to complete replication making a gap in the lagging strand

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20
Q

How is the gap in the lagging strand fixed?

A

The repair polymerase Dpol I, removes the RNA primer using its 5– 3 exonuclease action and simultaneously builds the new DNAstrand behind

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21
Q

A nick is left over after the dpol 1. How is this sealed?

A

DNA ligase

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22
Q

Initiation of E.coli - the oriC site contains a 9 mer region which is recognised by what?

A

DNAA- ATP initiator proteins

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23
Q

What does DNA a do when bound with ATP?

A

binds to 9 mer which can then bind to 13 mer which opens a small region of DNA

24
Q

What does opening of DNA allow?

A

DNAB to be loaded (main helicase)

25
Q

What happens after helicase is loaded?

A

a single stranded binding protein (SSB) binds on single strand to stop reannealing of strands

26
Q

After SSb binding what happens?

A

a DNA primase is added which synthesises short RNA primer

27
Q

After the primer is made what is added?

A

DNA polymerase 3 which starts elongation.

28
Q

In front of the opening there is DNA gyrase - what does this do?

A

helps deal with supercoiling changes

29
Q

Termination - they dont really know - what are terminus regions?

A

Sites that are bound by tus

30
Q

Termination - what does tus do?

A

When bound it can stop the replication fork from the opposite side, this is because the tus is stopping the strand whilst it waits for the strand on its own side (lagging) to meet it.

31
Q

What happens after the replication forks bump into one another?

A

the ribosome is removed and recQ and Topl II will get rid of peices of DNA not removed. The nick which is left is finished by DNA ligase.

32
Q

what happens when replication goes wrong - what is one way this can happen?

A

replication fork collapse

33
Q

What is replication fork collapse?

A

nicks or gaps in the DNA cause the fork to collapse as the polymerase meeting a single strand leads to a double strand break

34
Q

What happens if the replication fork collapses?

A

Potential loss of genetic information

35
Q

In a double strand break is the polymerase lots and the replication stop?

36
Q

How do you fix this double strand break?

A

RecBCD loaded onto the DNA and after chi site recognition loads RecA which looks for homologous region to invade and then forms a D loop.

37
Q

What happens to the d loop?

A

RUVABC translocated the Holliday junction and this is then cleaved creating a 3 way junction

38
Q

What is the 3 3 way junction recognised by?

A

replication restarts protein (PriA - restarts replication away from the origin site).

39
Q

How does the replication re-start work?

A

PriA recognises the 3 way junction and loads on it (this is a helicase) so opens the DNA before accessory proteins PriB, DnaT and DnaC. Once DnaC is loaded DnaB can then load as well (remember this is the key helicase for replication).

Replication

40
Q

What are the PriA targets?

A

D-loops - these are handled the same way

41
Q

Replication fork reversal and re-start - what causes replication fork reversal?

A

obstacles on DNA such as transcription collision, some replication defects, and strong binding of protiens on DNA

42
Q

What is replication for reversal dependant on?

A

RuvAB which facilitates reversal as they will recognise the 3 junction and want to anneal it into a 4 junction

43
Q

The basic idea of reversal replication forks is what?

A

They are converted into a 4 way junctions which is processed by a Holliaday junction.

44
Q

How is the Holliday junction formed in this case?

A

The newly synthesising strands can anneal to one another

45
Q

What happens after RuvAB is loaded?

A

RuvC recognises this and cleaves it = double strand break

46
Q

What happens to the double strand break in replication fork reversal?

A

It needs to be repaired

47
Q

Remember the lagging strand - what happens when there is a replication block?

A

There is a small area of single strands and RecA is loaded which helps the reannealing of the 2 strands. Then RuvAB binds

48
Q

What are some cases (DnaBts mutants) what is reversal dependant on?

49
Q

Can repairs proteins create breaks?

50
Q

After the RuvAB has created the 4 junction what could happen instead of RuvC celavage?

A

RecBCD could degrade DNA which would move the replication fork slightly backwards and priA would recognise it to initiation replication once more.

51
Q

What would happen if RecBCD didnt degrade all the DNA?

A

RecA would bind and then priA would recognise it.

52
Q

There is one more way replication can go wrong - this takes place at the Ter site, what is it?

A

Ter sites are blocked and another replication cycle is on the go - this means that the unfused original strands will run into the newly synthesised strands and form double strands. This is over replication

53
Q

What happens after the double strand fusion?

54
Q

What is the origin for replication arrest in fork reversal and the proteins involved?

A

Replisome transcription

RecBCD and PriA and its RecBCD dependant

55
Q

What is the origin for replication arrest in over replication and the proteins involved?

A

Specific collision at Ter sites
RecBC, RecA and RuvABC

56
Q

What is the origin for replication arrest in fork collapse and the proteins involved?

A

Nick or gap
RecBCD, RecA, RuvABC

Molecular Genetics (20 decks)

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