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Biochemistry 2 > DNA replication > Flashcards

DNA replication Flashcards

1
Q

what kind of cells undergo DNA replication

A

somatic cells (non germ cells)

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2
Q

basis for evolution

A

DNA is subject to changes that permit genetic differences

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3
Q

difference in DNA strands

A

polarity

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4
Q

DNA replication overview

A

strand separation followed by copying each strand

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5
Q

semi-conservative replication

A

strands separate, 1 parent, 1 daughter

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6
Q

what direction does DNA replication occur in

A

bidirectionally

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7
Q

where does DNA replication begin

A

at origins of replication

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8
Q

unwinding at double helix

A

1) helicases use ATP hydrolysis to separate strands (melt H bonds in btwn strands)
2) DNA gyrase creates negative supercoiling to overcome torsional stress that occurs during strand separation
3) single-stranded binding protein (SSB) binds to ssDNA so it doesn’t reanneal

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9
Q

DNAB helicase

A

binds to lagging strand

helicase in bacteria

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10
Q

topoisomerases

A

in euks = gyrases in bacteria

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11
Q

where are gyrases located?

A

ahead of replication fork

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12
Q

semi-discontinuous replication

A

Leading strand: continuous replication

Lagging strand: discontinuous replication

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13
Q

in which direction do DNA polymerases move

A

3’ to 5’

so since lagging strand is opened 5’ to 3’, there is slower replication

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14
Q 
summary
DNA gyrase
DnaB
DNA pol III holoenzyme
DNA pol I
A

DNA gyrase = unwinding DNA
DnaB = 5’ to 3’ helicase (DNA unwinding)
DNA polymerase III holoenzyme = elongation (DNA synthesis)
DNA polymerase I = excises RNA primer, fills in with DNA

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15
Q

DNA pol requires

A

all 4 dNTPs = substrate to make nucleotides
a template strand = substrate to catalyze next phosphodiester bond
primer = needed bc DNApol can’t start a new strand, only elongate one that already exists

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16
Q

what enzyme adds the primer to template strands

A

primase adds a short string of RNA nucleotides complementary to DNA template

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17
Q

processivity

A

ability of DNA pol to stay associated with DNA template (how many new phosphodiester bonds it can form)
different DNA pols have diff processivities

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18
Q

how do we pick the new bases

A

DNA pol uses base-pairing
new chain grows in 5’ to 3’
DNA pol can’t initiate a DNA strand, a 3’ OH is required

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19
Q

DNA pol 3’ to 5’ exonuclease activity

A

“proofreading”
If DNApol figures out it added the wrong base before it adds the next one, it can have 3’ to 5’ exonuclease activity to remove the wrong base
uses a couple ATP

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20
Q

polymerase catalytic sites

A

1) polymerase site (makes new phosphodiester bond) 2) 3’ to 5’ exonuclease hydrolysis site

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21
Q

DNA pol II and II (E. coli)

A

3’ to 5’ exonuclease activity

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22
Q

DNA pol I (E. coli)

A

3’ to 5’ and 5’ to 3’ exonuclease activity

it can remove and replace nucleotides concurrently

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23
Q

E.coli DNApol

A

pol I, II and IV involved in DNA repair
pol I also helps with replication
DNA pol III is the replicating enzyme

24
Q

DNA pol structure

A

claw shape
Fingers (alpha helices): recognizes dNTPs
Thumb: DNA binding
Palm: contains the polymerase active site

25
Q

WHY DNA POL ONLY WORK IN 5’ TO 3’ DIRECTION?

A

Release of pyrophosphate provides energy to form new bonds
The pyrophosphate is released from the template strand (from incoming dNTP) when we go 5’ to 3’ → if a mistake is made, we lose a dNTP, but we have tons around
If dna pol went other way, pyrophosphate would come from growing strand
If a mistake → energy is spent and we couldn’t repair bc we lost energy
Once phosphodiester bond is made, we don’t have energy for repair

26
Q

WHY DO WE NEED AN RNA PRIMASE TO MAKE PRIMERS?

A

RNA polymerases can bind 2 nucleotides at the same time and can make phosphodiester bond between 2 NTPs
DNA polymerases can’t start a strand

27
Q

DNA pol III

A

multi-subunit holoenzyme
core: alpha, epsilon, theta
other key subunits: tau, beta, gamma, delta, delta’

28
Q

DNA pol III beta

A

Beta is sliding clamp that surrounds DNA and holds core enzyme onto ssDNA
Responsible for high processivity of DNA pol III

29
Q

DNA pol III core enzyme function

A

exonuclease and polymerase activity
alpha, epsilon, theta
synthesizes lagging strand

30
Q

gamma complex

A

made of one gamma, one delta, one delta prime
clamp loader
loads beta clamp onto DNA
(uses ATP hydrolysis for energy to assemble a pair of beta subunits on to each DNA strand at replication fork)

31
Q

tau complex

A

holds 2 cores together

32
Q

how is DNA pol III removed

A

core polymerase synthesizing lagging strand must be released at end of each Okazaki fragment
beta-clamp is released by tau and is reloaded using gamma complex onto next Okazaki fragment

33
Q

2-beta subunits =

A

beta clamp

34
Q

DNA pol III at replication fork

A

both strands copied at same time (coordinated by presence of 2 DNA pol II complexes at replication fork

DnaB also associates with these complexes

35
Q

in which direction do both strands elongate

A

5’ to 3’

36
Q

DNA ligase

A

seals gaps between Okazaki fragments

37
Q

Tus proteins

A

required for termination of DNA replication

38
Q

starting and stopping DNA replication in E. coli

A

opposite or origin (ori) is terminator (ter) sites)
ori is AT rich
terminator sites binds to Tus proteins
Tus proteins block replication in one direction (each set (group 1 or 2) blocks in one direction)
terminator = replication machinery releases template strand

39
Q

replicons

A

replication factories
DNA pols are in fixed locations (attached to a membrane)
DNA is fed through polymerases
new and parent strands emerge from factories as loops

40
Q

DNA replication in euks

A

Occurs only during discrete phase of the cell cycle (S for synthesis)
Progression through the cell cycle is regulated by cyclins and CDKs
Cyclins are produced in a phase-specific manner
Cyclins activate CDKs that stimulate phase specific events

41
Q

which phase must DNA replication occur in?

A

S phase ONLY

42
Q

origins of replication in genome

A

Multiple origins of replication in the genome
Each replication origin fires only once per cell cycle
Licensing occurs in G1, activation occurs in S

43
Q

licensing of DNA replication

A

requires assembly of pre-replication complexes at each origin of replication (late M or early G1)
ORC binds each origin
ORC recruits Cdc6p, Cdt1, and MCMs (one is a helicase)

44
Q

with start of S-phase,

A

S-cyclin binds to its CDK and activates kinase activity
S-CDK phosphorylates its targets
phosphorylation of MCM proteins activate helicase and recruits Cdc45 and a primase

45
Q

how many replication forks?

A

2: bidirectional replication

46
Q

cyclin licensing: when is it bad?

A

we need to prevent cyclins from being licensed again in S phase (this would cause another round of replication)
Geminin: prevents licensing in S phase

47
Q

Geminin

A

targets MCM proteins and prevents and prevents them from binding to ORC
Geminin degrades in M phase

48
Q

DNA pol alpha

A

has primase subunits

RNA + DNA

49
Q

PCNA

A

analogous to beta clamp in bacteria
It is made of 3 subunits instead of 2, but serves the same function
Replication factor C (RFC) loads the PCNA clamp onto replicating DNA
RFC is analogous to gamma clamp loader

50
Q

end replication problem

A

DNA pol requires a 3’ OH on the previous nucleotide to extend the strand following excision of RNA primers
At the extreme ends of each strand, no such 3’ OH exists
Once RNA primers are removed, there’s no 3’ OH
The RNA primers at the 5’ ends of each newly synthesized strand CANNOT be replaced by DNA bc DNAP can only extend a strand

51
Q

what happens with each round of replication

A

Anytime somatic cells replicate, they lose a bit of DNA
Telomeres help protect ends
Telomerase builds telomeres

52
Q

telomeres

A

GC rich repeat sequences

telomerase replicates telomeres and contains enzyme called TERT and RNA template

53
Q

why do we need telomeres

A

at end of replication, RNA primers are removed and must be replaced with DNA
this leaves a gap at extreme 5’ end of each strand
telomerase uses RNA template which hybridizes with end of completed strand
this provides template to extend 3’ end of top strand
bottom strand is then synthesized

54
Q

reverse transcriptase (RT)

A

makes DNA using ssRNA as a template
Primer is dNTP from host that binds with ssRNA → forms helix with 1 ssRNA and 1 ssDNA
RNase H takes RNA out and leaves us with ssDNA
AIDS drugs are nucleotide analogs that block RTase activity

55
Q

telomeres are rich in

A

GC

sequence depends on species

56
Q

why do we need TERT?

A

DNA polymerase can’t extend both ends bc it requires DNA as a template
TERT uses RNA as a template

Biochemistry 2 (32 decks)

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