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Flow of genetic information BIOL2010 > DNA replication > Flashcards

DNA replication Flashcards

1
Q

How does DNA polymerase I work and what is it?

A

first enzyme to be isolated by Kornberg in 1958.
is a single polypeptide chain and there are around 400 of them per cell.

it requires all 4 dNTPs, a template strand and a primer.

it isn’t self priming as it can only ad onto existing 3’-OH terminus.

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2
Q

Where does Pol I bind to on DNA?

A

binds to nicked or gapped DNA.

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3
Q

What direction is chain growth in polymerisation?

A

grows in 5’ to 3’ direction.

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4
Q

What are some of the activities of POL I

A

5’-3’ polymerase
3’-5’ exonuclease (proof reading)
5’-3’ exonuclease ( nick translation)

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5
Q

What is produced if a POL I is treated with proteases?

A

get small N terminal fragements (5’-exonuclease)

large C terminal fragments (Klenow fragment) - contains polymerase and 3-‘ exonuclease.

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6
Q

what is the mechanism of Klenow fragments?

A

the Klenow fragment requires two bound metal ions (Mg2+) held in place by aspartate residues.

it positions the 3’ end of the primer and incoming dNTP to enhance catalytic reaction.

its shape is complementary to the correctly form base pair leads to specificity as the wrong paring wont be able to fit through the fragment correctly as wrong pairings are the wrong shape and size.

retains polymerase and 3’ to 5’ exonuclease to remove 3’ overhangs or fill in 5’ overhangs to form blunt ends.

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7
Q

How does the Klenow fragment know which nucleotide to add?

A

formation of correct H bonds.

shape as incorrect base pairs wont fit into the helix.

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8
Q

What is the main eukaryotic DNA replication enzyme?

A

DNA POL III

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9
Q

Features of POL III?

A

only 10 per cell. has a high rate of polymerisation around 1600 nucleotides per second.
is highly pocessive and is a complex multimeric enzyme.

polymerases in 5’-3’ and 3’-5’ exonuclease activity for proof reading.
it does not contain 5’-3’ exonuclease activity for nick translation.

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10
Q

How can we determine which enzymes (proteins) are important in DNA replication?

A

use temperature sensitive mutants. divided into two broad categories

quick stop mutants that immediately stop DNA replication
slow stop mutants that allow replication to finish but cant start another round.

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11
Q

Why are there so many enzymes in DNA replicaiton?

A

DNA strands are antiparralel

all polymerases are 5’ to 3’ and are not self priming so need a primer

DNA strands are plectonemically coiled and need to be physically separated for semi conservative replication (e.g with DNA helicase)

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12
Q

How is the lagging strand of the 3’ to 5’ direction synthesised?

A

made up in pieces called Okazaki fragments. they are then joined together via DNA ligase. (requires ATP or NAD to join the nicks)

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13
Q

why does Uracil appear in DNA?

A

gets incorporated in place of T but is non offensive as has the same base paring as T. Can also appear from the spontaneous deamination of C but this is offensive as makes a GU pairing in place of GC which causes mutation to AT in the next round of replication.

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14
Q

how is Uracil removed form DNA? why did Okazaki think both strands were discontinuous?

A

gets removed with uracil-N-Glycosylase and it removes U opposite G but does not discriminate against U opposite A. during replication temporary nicks are created making pseudo Okazaki fragments on the leading strands.

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15
Q

How is proof reading done?

A

strand extension at the 3’ end only proceeds if the correct base has been added if not then the 3’exonuclease activity of pol III (dnaQ) removes it.

dnaQ - mutants accumate errors.

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16
Q

What syntheses primers and why are they needed?

A

synthesised by primase (dnaG). required to allow DNA polymerases to extend onto the 3’-OH end.

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17
Q

What is primase?

A

an RNA polymerase that is self priming and works in 5’ to 3’.

has no self editing functions such as proof reading and tend to be around 5- 10 nucleotides long.

activity of the enzyme is increased by the presence of a helicase.

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18
Q

How can we tell that primase is an RNA polymerase?

A

When Okazaki fragments are treated with RNase and then centrifuged get an intermediate band between RNA and DNA showing that the fragments are made of DNA and RNA (from the primers)

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19
Q

before Okazaki fragements are joined together via DNA ligase what occurs to the RNA primers?

A

POL I removes the primers and RNA and then DNA ligase cans seal the gap. (Nick translation)

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20
Q

What gene produces DNA helicases

A

dnaB

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21
Q

how does the DNA fork get unwound?

A

gets opened up and unwound by DNA Helicase. its a DNA dependant ATPase that acts progressively moving along DNA form 5’ to 3’ on lagging strand

22
Q

what is a condition that occurs due to defective helicases?

A

Blooms and Werner syndrome. Causes premature ageing.

23
Q

what are single stranding binding proteins?

A

proteins that stabilise the single strand of unwound DNA. Ssb is the main product of the dnaT gene.

binds cooperatively mainly on the lagging strand. binding of one enhances the binding of another ssb.

needed so the unwound dna doesnt start to ravel up and create hairpins and loops.

24
Q

What is the beta clamp of the POL III holoenzyme for and how does it reach the DNA?

A

beta clamp forms a ring around the DNA to ensure processivity and it reaches the first by the clamp ladder which is a multisubunit that uses ATP to load it.

25
Q

What is a topoisomerase?

A

are enzymes that releive the superhelical stress that is produced in front of and behind the replication fork. they also have the road of seperating the two daughter DNA molecules after the cycle of DNA replication is complete.

26
Q

What is the linking number in supercoiling?

A

it is the number of times that one strand passes around the other. for a circular genome the linking number cannot be changed unless the circle is broken and then either wound more or less. (overwound or underwound)

27
Q

what is twist and writhe in supercoiling?

A

the twist is the number of helical turns in the DNA and the writhe is teh number of times the double helix crosses over itself. the linking number is the sum of these.

28
Q

Topoisomerase’s alter linking number. T/F

A

True

29
Q

What is a type 1 topoisomerase and how does it work?

A

topoisomerase III is a type 1 and it catalyses the relaxation of the supercoiled DNA. is thermodynamically favoured. decreases linking number by 1.

30
Q

What is type 2 topoisomerases and how do they work?

A

topoisomerase IV which is a type 2 introduces negative supercoils to DNA using free energy from ATP hydrolysis.

they cut both strands of a duplex and pass one strand through the other. decreases L by 2

31
Q

What is the structure of DNA gyrase and what is it?

A

it is a prokaryotic enzyme not involved in replicaiton. it introduces negative supercoils (type 2 topoisomerase) and requires ATP. Decreases L by 2 at a time and has an A2B2 structure.

32
Q

Where does initiation and termination of replication occur?

A

at fixed OriC sequences. from here the replication fork moves bidirectionally until reaches the termination sequence TerC

33
Q

What is the sequence for OriC and how mant base pair repeats does it have?

A

Is 245 bp sequence. has 4x9 bp repeats and 3x13 bp repeats that are both AT-rich.

34
Q

What protein is specific for the initiation of replication?

A

DnaA. is a replication initiation factor that promotes the unwinding of DNA at OriC.

35
Q

WHy does initiation only occur once per cycle?

A

OriC contains lots of GATC sequences that are the substrate for DAM (DNA adenosine methylase)

DAM methylates N6 of adenine which immediately after replication become hemimethylated. hemimethylation inhibits initiation.

36
Q

What does protein Tus do that terminates replicaiton?

A

the binding of Tus to the Ter region (termination) prevents fork movement in one direction only.

37
Q

What is the Ter region?

A

the site of termination. contains 6 homologous 23 base pair sequences. has 3 sites in each direction to bind to Tus.

38
Q

What seperates two circular daughter chromosomes?

A

Topoisomerase IV.

39
Q

What are some of the differences of DNA rep in eukaryotes compared to prokaryotes?

A

occurs in the nucleus not cytoplasm.

more genetic material to replicate

DNA is more than 1 chromosome.

not circular

additional packaging (histones, nucleosomes etc)

40
Q

in terms of replication forks what is different between prokaryotic and eykaryotic rep?

A

prokaryotes replicate from a single origin. in eukaryotes there are 1000s of replication forks. these are from ARS (autonomously replicating sequences)

41
Q

compare prokaryotic and eykaryotic okazaki fragments

A

eukaryotic okazaki fragments are much shorter (about 135 bp long)

42
Q

what is the sliding clamp in eukaryotic replicaiton known as?

A

proliferating cell nuclear antigen. (PCNA)

43
Q

Why is polymerase alpha not very processive?

A

it has its own primase activity but it is not very processive as it does not assoicate with PCNA and does not have a 3’-5’ exonuclease.

44
Q

what are polymerases delta and epsilon

A

pol delta associates with PCNA and does processive synthesis (like POL III)
pol epsilon displaces the primers (analogous to POL I)

45
Q

In eukaryotic rep what removes the RNA primers and why?

A

FEN1 (flap) exonuclease removes the RNA primers and this is because eukaryotic polymerases do not have a 5’ -3’ exonuclease.

46
Q

what is an ORC an what are they for?

A

Origin Recognition Complex an they bound to the DNA at each replication origin.

47
Q

Why do chromosomes shorten after each round of replication?

A

Rimer primers are removed by nick translation EXCEPT the first one. so no replication is possible at the first one. thus each round the chromosome gets shorter.

48
Q

how is the end replication problem solved in eukaryotic rep?

A

addition of repeated units of simple sequences to the chromosomal ends (telomeres). they are added DIRECTLY by the enzyme telomerase.

49
Q

what regulates telomere length?

A

it is regulated by the telomere bidning proteins TBP such as TRF1 an TFR2.

50
Q

What polymerase copies the RNA genomes of animal and plant viruses?

A

RNA replicase.

51
Q

Why do RNA genomes not need primers or exonuclease?

A

they are self priming and dont have proof reading activty thus are highly error prone?

52
Q

What enzyme can prepare a viral genome to be intergrated into a host genome?

A

Reverse transcriptase take the single stranded RNA of the virus and double it into DNA which is double stranded.

Flow of genetic information BIOL2010 (4 decks)

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