DNA Replication Flashcards Preview

MoMF > DNA Replication > Flashcards

Flashcards in DNA Replication Deck (37):
1

Aicardi-Goutieres syndrome

Cerebrospinal fluid has elevated white cells (lymphocytes) and high levels of interferon-alpha (INF) activity
Atrophy in frontal and temporal regions of brain
Followed by loss of almost all motor skills

2

Initiation- prokaryotic replication

One start site for DNA replication --> initiation site
In ecoli: oriC

Initiator protein + ATP + histone like proteins attach to oriC causing DNA to wrap around the complex creating negative superhelical tension in the adjacent DNA

Results in the opening of the double helix at 3 x 13 AT rich repeats left to the protein complex

Further opening the DNA creates a replication bubble

3

oriC

oriC has 4 sequences 9 bp in length that are recognized by the initiator protein

Adjacent to oriC are 3 AT rich repeats

4

Why are AT rich repeats important?

Provides a site for DNA to open easily (more unstable than GC rich DNA)

5

How many replication forks per replication bubble?

2

6

What are the limits of DNA polymerase?

DNA polymerase cannot initiate DNA synthesis (new strand synthesis)

Requires a 3' hydroxyl group at the end of a base paired strand to add dNTPs

7

RNA primers

synthesized 5' --> 3' and are laid down antiparallel and complementary to the DNA template strands (both stands of the parent DNA)

Reaction is catalyzed by primase

8

primosome

primase+ helicase + protein

binds both strands of the replication bubble and makes a short RNA primer

9

DNA polymerase 1

RNA primer excision
DNA repair
3' and 5' exonuclease activity

not the main synthetic enzyme in E coli

Processivity: 3-200

10

3' exonuclease activity

when an incorrect base is incorporated the 3' exonuclease removes it, correct base can be inserted

11

5' exonuclease activity

removal of RNA primers in Ecoli

12

DNA polymerase 3

synthesizing enzyme in replication- replicative chain elongation

processivity- 500,000

functions as a holoenzyme, with a core of 3 polypeptides

13

Core of DNA polymerase 3

alpha- dimer polymerase
epsilon- dimer 3' exonuclease for proofreading
beta- dimer that forms sliding clamp around DNA

*dimer so that it can replicate both strands of DNA at the same time*

14

beta dimer in pol III

internal diameter of 3.5 (just enough to fit 2 nm diameter for DNA) that has a low affinity in its inner surface to DNA so that the DNA ca slide smoothly along it

15

Gamma complex of DNA polymerase 3

made up of various holoenzymes that help the beta subunit unload onto the DNA --> clamp loading

16

Chi and Psi subunits of DNA pol 3

Also bind to the g complex

Chi subunit mediates transition from synthesizing RNA primers to DNA

17

T complex DNA pol 3

Ensures that the core enzyme is a dimer, allowing both strands of the replication fork to be synthesized simultaneously

also binds to helicase

18

Lagging strand synthesis

Has to go from 5 --> 3

the fork has to open up BEFORE new synthesis can be initiated

19

What is the function of the loop?

Allows DNA synthesis to run smoothly and in the same direction for both strands

lagging strand folds to make a small loop, this allows the lagging strand to be in the same orientation as the leading strands and now they can be copied together by the holoenzyme dimers.

20

What is formed at the end of DNA replication for bacteria?

2 lagging strands and 2 leading strands of new DNA

21

Termination- prokaryotic replication

termination region is 180 degrees from the oriC (ter region)--> 20 bp segment

when replication fork meets TUS-ter complex, it halts...ensuring that each of the two forks initiated at oriC will travel no farther than ter

22

Proofreading

DNA Polymerase III can recognize mismatched base pairing (mainly AG and CT)

The epsilon subunit removes the mismatched base pain with its 3' to 5' exonuclease activity

The chain is then extended by DNA polymerase III

23

Nick Translation

DNA polymerase I 5' to 3' exonuclease hydrolyses the RNA primers

Simultaneously, the 3' end of the Okazaki fragment (DNA) is extended by incorporation of deoxyrobonucleotides

DNA pol 1 moves to replace the RNA fragments in the previous Okazaki fragment with DNA, the "nick" keeps moving over one space until the end where ligase seals it together

24

Final step in termination of DNA synthesis in E coli

type II topoisomerase separates the 2 interlinking strands of the replicated circles and refold the DNA into supercoils

25

DNA polymerase α

Found in nucleus
Has primase activity --> can make RNA primer in lagging strand replication
Moderate inherent processivity
Moderate processivity with PCNA (like a clamp)

26

DNA polymerase δ

Found in nucleus
Lagging strand synthesis
Low inherent processivity
High processivity with PCNA
3' exonuclease activity

27

DNA polymerase ε

Found in nucleus
Leading strand synthesis
High inherent processivity
High processivity with PCNA
3' exonuclease activity

28

DNA polymerase γ

Found in mitochondria
mitochondrial DNA replication
High inherent processivity
High processivity with PCNA
3' exonuclease activity

*not sensitive to aphidicolin

29

Sensitivity to aphidicolin (fungal steroid that inhibits replication)

To see which specific polymerases were associated with replication

30

3 prime structure in replication

Nucleosomes are dissociated ahead of the replication fork, and reassembled on newly synthesized DNA

31

Okazaki fragments in eukaryotic DNA replication

Size is much smaller than in prokaryotic synthesis

32

Telomeres

Highly repetitive sequences at the 3' ends of linear chromosomes

33

What is added by telomeres?

Protein component enzyme- reverse transcriptase- hTERT (human telomerase reverse transcriptase"

RNA component- hTR (human telomerase RNA)
Template

34

Why do we need telomeres?

When you come to the last primer in the lagging stranding, polymerase 1 cannot complete gap at the end --> need OH group for DNA pol to work

35

How does telomerase work?

Telomerase uses RNA component to add repeats

Telomerase repositions itself to add each repeat (moves 3' --> 5' on daughter strand)

The daughter strand is synthesized using complementary base pairing by DNA polymerase alpha

This process is repeated ultimately forming the long telomere

36

3' overhang at telomere end

A single stranded 3' overhang left when telomerase moves away

37

D-loop T-loop

Telomere ends are protects by loops that are stabilized by binding of telomere-binding proteins called TTAGGG repeat binding factors