DNA replication

Question 1

outline briefly gene expression?

Answer 1

1. transcription - nucleotide sequence of gene copied into RNA
2. translation - nucleotide sequence of RNA directs order —> AA —> protein

Question 2

describe the Hersey Chase (1952) experiment?

Answer 2

tried to prove dna was genetic material

• viruses grown in one of two isotopic medium to radioactively label specific viral component
• virus gown in radioactive sulphur (35S) had radio labelled proteins (sulphur present in proteins NOT dna)
• virus grown in radioactive phosphorous (32P) had radio labelled dna (phosphorous present in dna not proteins)
• virus then infected bacterium (e.coli)
• virus + bacteria separated by centrifugation
• larger bacteria = solid pellet
• smaller viruses left in supernatant

Question 3

what did the Hersey Chase experiment prove?

Answer 3

• bacterial pellet found = radioactive when infected by 32P-viruses (DNA) + NOT 35S virus (protein)
• DNA NOT PROTEIN = genetic material
• DNA transferred to bacteria

Question 4

what does semi-conservative dna replication give?

Answer 4

2 duplex DNA mol each of which contain one old + one new strand

Question 5

what does conservative dna replication give?

Answer 5

2 duplexes

one has 2 old strands and second has 2 new strands

Question 6

what does dispersive dna replication give?

Answer 6

2 daughter duplexes

both contain mixture of old and new strands

Question 7

experiments by meselsohn + stahl (1958) proved which dna replication model?

Answer 7

semi - conservative

Question 8

describe the meselsohn + stahl experiment?

Answer 8

• grow bacteria in 15N (heavy) medium
• transfer some bacteria to 14N (light) medium —> bacteria grows
• take samples after 0, 20 and 40 mins
• before bacteria reproduce for 1st time in light medium (0mins), all DNA (parental) = heavy
• after 2 gen, half dna = intermediate and half = light only (no only heavy dna)

Question 9

how long is one round + 2 rounds of replication?

Answer 9

one round = 20 mins

two rounds = 40 mins

Question 10

what was the conclusion obtained from the meselsohn + stahl experiment?

Answer 10

pattern only observed if each dna molecule has template from parental dna —> dna replication = semi-conservative

Question 11

replication is initiated at an ‘origin of replication’ that gives rise to two…

Answer 11

replication forks

Question 12

what is a replication fork?

Answer 12

branch point in replication eye at which DNA synthesis occurs

Question 13

what may a replication bubble contain?

Answer 13

1/2 replication forks (unidirectional/bidirectional replication)

Question 14

what is dna replication almost always?

Answer 14

bidirectional

Question 15

how many replication origins do prokaryotic and bacteriophages have?

Answer 15

1

Question 16

what did reniji Okazaki elucidate?

Answer 16

semi-discontinuous model of dna replication

Question 17

prokaryotic replication initiation - dnaA proteins?

Answer 17

• 4 copies of 9-bp sequence that bind dnaA proteins
• once all binding sites full ——> cooperatively recruit more dnaA proteins = dnaA barrel
• helps open up local AT rich region of dna via TORSIONAL STRESS
• pair of replication forks generated

Question 18

prokaryotic replication initiation - dnaB proteins?

Answer 18

• dnaB recruited to replication fork to initiate formation of pre-priming complex
• dnaB = helicase enzyme: breaks HB
• open dna strands covered with SSBs (single stranded binding proteins)
• SSBs stop strands re-annealing + protect dna from attach by free radicals + nuclease enzymes
• initiation complete
• next phase = elongation

Question 19

describe the polymerase reaction?

Answer 19

synthesis of new strand carried out by dan-dependent DNA polymerase enzymes

• dna generated in 5’ to 3’ direction
• polymerase moves along template strand in 3’ to 5’ direction

Question 20

describe prokaryotic elongation?

Answer 20

• primase enzyme (DnaG) binds near to heliase
• starts to synthesise RNA primer on primosome
• single stand binding protein stabilise lagging strand
• dna polymerase III holoenzyme clamps to leading strands + synthesises dna

Question 21

semi-discontinuous replication

Answer 21

carried out by dna polymerase in 5’ to 3’ direction

• polymerase enzyme inserts 5’ nucleotide first + extends towards 3’ end

template dna mol always used in 3’ to 5’ direction

Question 22

what is the lagging strand generated via?

Answer 22

synthesis of multiple Okazaki fragments

Question 23

the lagging strand is generated in ….. direction to movement of replication fork

Answer 23

opposite

Question 24

all dna replication is started with a short rna primer.

what does this allow?

Answer 24

proofreading of newly synthesised strand

Question 25

how are primers removed?

Answer 25

by the exonuclease activity of the polymerase complex

Question 26

what allows both strands of dna to be replicated at the same time?

Answer 26

2 dna polymerase enzymes = tethered together - each replicating one strand

Question 27

what is a replisome?

Answer 27

leading strand continuously in 5' to 3' direction lagging strand discontinuously in 5' to 3'

Question 28

describe the nature of the lagging strand?

Answer 28

looped over top of replisome so both polymerases move in same direction

Question 29

which enzyme proceeds ahead of the replisome?

Answer 29

topoisomerase

Question 30

how are the Okazaki fragments joined?

Answer 30

by ligase enzymes (ligation) - dna polymerase III replaced with another polymerase (pol I) - removes rna primer on Okazaki fragment before ligation

Question 31

why are topoisomerases needed to alleviate the problems within dna elongation?

Answer 31

- replication fork can only progress short distance before there = topological prob - double helix needs rotation as helix = opened to stop over-winding ahead of replication fork

Question 32

what are the 2 types of topoisomerases?

Answer 32

Type I - introduces real in 1 strand —> pass other strand through + then reseal break Type II - breaks both strands —> passes double helix through gap —> reseals break

Question 33

how the breaks in dna attached and why?

Answer 33

covalently so ends don't loosen

Question 34

in e.coli where do the 2 replicons meet?

Answer 34

180 degrees away from the origin

Question 35

what is in place to make sure the replicon meed at a specific point?

Answer 35

regulatory mechanism if 1 gets there first, it will wait for the 2nd to arrive before signalling completion of dna replication

Question 36

what do specific terminator sequences signal?

Answer 36

that replication is approaching stop sequence

Question 37

what happens if the replicon meets a transcription bubble (mRNA synthesis)?

Answer 37

will wait and not overtake

Question 38

describe eukaryotic replication forks?

Answer 38

- slower than prokaryotic forks - move at around 50bp per second - multiple forks needed due to size (50,000-100,000) per mammalian cell

Question 39

in eukaroytes, clusters of how many replicons will initiate simultaneously at defined time in S-phase based upon mitogen availability?

Answer 39

20-50

Question 40

early S-phase clusters

Answer 40

euchromatin- transcriptionally active dna

Question 41

late S-phase clusters

Answer 41

heterochromatin-transcriptionally silent dna

Question 42

centromeric and telomeric dna

Answer 42

replicated last

Question 43

how is the sequence in yeast bound?

Answer 43

- by origin replication complex (ORC) - activated by cyclin dependent kinases (CDKs) - facilitates/initiates opening of dna duplex

Question 44

how many times can eukaryotic dna replicons initiate?

Answer 44

once per cell - so dna = fully + controllably replicated before cell division - limits introduction of mutations

Question 45

what is needed for the initiation of dna replication?

Answer 45

- protein licensing factor complex - inactivated after use - only able gain access to nucleus when nuclear envelope dissolves in mitosis

Question 46

basically the origin is..

Answer 46

- identified (binding of ORC) - set up (binding of initiation factors) - checked - initiation

Question 47

why can the ends of chromosomes not be replicated by semi-discontinuous replication?

Answer 47

no dna to elongate once rna primer removed from 5' -end of lagging strand could lead to loss of genetic material

Question 48

how can u overcome this problem?

Answer 48

eukaryotic chromosomes have hundreds of copies of non-coding repeat sequence 3' end hangs over 5' end telomerase associates with short rna mol that = partially complementary to this sequence rna acts as template for addition of repeats to 3' end overhangs comp strand = synthesised by normal lagging strand synthesis —> leaves 3' overhang

Question 49

where is telomerase activity repressed and why?

Answer 49

- in somatic cells leads to gradual loss of dna + shortening of chromosomes may cause problems in ageing process