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Molecular Biology BIOL10221 > Module 2 - DNA replication > Flashcards

Module 2 - DNA replication Flashcards

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1
Q

why is DNA replication important

A

DNA needs to double to pass on equal amount of DNA

essential for reproduction and continued life

defects in DNA rep can cause disease (e.g. cancer)

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2
Q

general idea of DNA replication

A
  • DNA strands seperate
  • DNA copied using parent strand as template
  • comp base pairing
  • semi cons replication
    = forms 2 identical strands same as parent DNA
    each daughter strand has half a strand from parent
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3
Q

other than semi conservative, what other ways could DNA possibly replicate

A

Conservative - one fully original, one fully new
Dispersive - both strands formed are a combo of old and new

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4
Q

Conservative replication

A

In theory:
- semi cons rep occurs
- 2 strands that are half old half new are made as per usual
- but then some kind of detachment and reassociation occurs where:
old strands combine w/ each other
new strands combine w/ each other

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5
Q

Dispersive replication

A

in theory:
two strands seperate
as the new strand moves along the template strand, the template switches repeatedly
- creates daughter molecules that are a combo of old and new

in 1950s, this was what most accepted

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6
Q

why did scientists think semi cons was impossible?

A
  • DNA is plectonemic = strands cannot seperate without unwinding
  • and cuz many DNA are circular, so cannot theoretically unwind
    e.g. bacteria DNA, mitochondria DNA, plasmids etc.

unless you cut it
BUT
- bacteria chromosome is ~4mill bp
- if this was converted to linear wouldnt work theoretically cuz
- bac divide every 20 mins
- so must replicate 200,000 bp per min (10bp per turn of helix)
- so 20,000 revs per min

so impossible, will pretty much explode

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7
Q

Explain how Meselson - Stahl experiement proves semi cons

A

Cultured E.coli bacteria in medium with HEAVY isotope of NITROGEN (15^N) using heavy NH4CL (ammonium chloride just in case u forgot basic chem)

N is component of DNA bases
so gets used by bacteria in their DNA

so to distingush between DNA w/ heavy or lght nitrogen,
= density gradient centrifugation
50,000x for 48hrs
- low dens at top
- hevy dens at bottom
due to BUOYANT DENSITY

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8
Q

explain method of mehselon-stahl’s experiement

A

E.coli cultured in heavy NH4Cl

transferred to light NH4Cl culture

grow for diff amounts of time:

  • after 20mins, isolate dna (=one cell division
  • do dens gradient centrifugation
    = ended up with intermediate band (so half and half)
  • after 40mins, isolate dna (=two cell divisions)
  • do dens grad centrifugation
    = ended up with one intermediate (same as prev)
    and one light (two strands of dna both with light N cuz thats what it was cultured in)
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8
Q

what are the predicted bands for all types of replication

A

Conservative =
after 1 rep
1 molecule with all heavy
1 molecule with all light

Dispersive =
after 1 rep
exact same as semi cons (cuz has an intermediate strand)
BUT
after 2 rep
stays intermediate (so stays as one intermediate band)

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9
Q

so how tf does circular DNA go through semi cons replication?

A

TOPOISOMERASES hehheh

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10
Q

Role of topoisomerases

A

prevents supercoils during replication

supercoils - get super knotted and replication cant go on with them

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11
Q

Types of DNA topoisomerase: TYPE 1

A

TYPE 1 DNA topoisomerase:
- ‘nick’ a strand, moves the other strand through the nick, gets rid of knot by ‘removing a helix’
- opens up structure
look at diagram if ur lost idk how else to explain it

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12
Q

Types of DNA topoisomerase: TYPE 2

A

TYPE 2 DNA topoisomerase:
major enzyme
- so there’s two segments of DNA supercoiled
- cuts BOTH strands (so like cuts through whole thing)
- G segment = DNA strand that gets cut so other strand can pass through
- T segment = DNA strand that gets passed through

see diagram in onenote its clearer

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13
Q

Why are DNA polymerases important

A
  • cellular enzymes that make DNA strands from nucleotides
  • mutations in these can cause colorectal cancer
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14
Q

what do DNA helicases do

A

break aprt the base pairs by hydrolysing the H bonds

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14
Q

how is DNA synthesised at the replication fork and what is the process called?

A

Template-dependant DNA synthesis

15
Q

what direction does DNA synthesis go in and what is required to initiate synthesis?

A

5’ to 3’
a primer is required

16
Q

What is exonuclease activity

A

DNA polymerases degrading DNA
2 types of exo nuclease activity:

3’->5’
basically means if theres a mistake in the nucleotide, it can reverse itself and go back to correct it
this is called PROOFREADING

5’ to 3’
can displace DNA that’s ahead of it

17
Q

all DNA polymerase types involved in replication

A

see table in onenote

18
Q

how are the seperated single strands protected

A

Single-strand binding proteins (SSBs)

  • prevents them reattching to each other
  • prevents them being attacked by nucleases

e.g. in eukryotes = replication protein A

19
Q

if one strand is the template strand, what’s the other one called?

A

leading strand - has continuous synthesis

20
Q

what is the primer made out of

A

made of RNA by the primase enzyme
it’s 4-15 nulceotides in length
once primer comes in, DNA pol III makes the new strand

21
Q

process in eukaryotes

A

sry for the poo questions idk how to word it
- RNA primer extended by DNA pol ALPHA
- alpha adds about 20 nucleotides
- then DELTA makes the rest of the strand

BECAUSE
alpha doesn’t have the 3’->5’ exonuclease mechanism that is required for proof reading
so it switches to delta

22
Q

DNA synthesis at site

A

beta and gamma phosphates are removed from nucleotide
on one end alpha phos binds to the sugar on the previous nucleotide AT 3’ OH terminus
bases join together on the other end

see diagram

23
Q

lagging strand and Okazaki fragments

A

because DNA synth always has to be 5’ -> 3’
lagging strand is on the other side
and because synthesis always starts with a primer
it has to be made in sections
sections = Okazaki fragments
these must be joined together and RNA primers must be removed
2 ways to remove RNA primers, in bacteria and in wukaryotes

24
Q

in lagging strand in bacteria, how are the Okazaki fragments combined?

A

when the DNA pol III reaches the primer, it stops and switches to DNA pol I, then continues to synthesise

cuz
DNA pol III has no 5’->3’ exonuclease activity
and DNA pol I does

then, DNA ligase ‘glues’ the two ends of DNA fragments together

25
Q

in lagging strand in eukaryotes, how are the Okazaki fragments combined?

A

DNA pol delta + helicase push the primer aside
FEN1 (an ENDONUCLEASE) cuts at the branch point
DNA ligase links the fragments

cant just get rid of primer CUZ
non of the eukaryotic polymerases have 5’-3’ exonuclease

26
Q

why in a linear molecule, would the dna molecules get shorter and shorter gradually

A

the final okazaki fragment can’t be made
priming site would be after the end of the parents molecule
so it would just cut short
hence why we need telomerase

27
Q

what does telomerase do

A

extends the parent DNA by adding TTAGGG several times so the final okazaki fragment can be primed
almost like a buffer zone

28
Q

what is telomerase made of

A

is an RNA/protein complex
few thousand telomere repeats at the end of human chromosoms

29
Q

what is the name and properties of the organism that has the telomere sequence of TTGGGG

A

Tetrahymena thermophilia
single cell ciliated organism
has 40,000 chromosomes
obvs needs lots of telomerase
used in discovery of telomerase cuz theres so much

30
Q

which cells have telomerase

A

only stem cells

or some cancer cells - which is why they divide continuously

31
Q

what is senescence

A

cells die
can divide ~50x
becuase normal cells dont have telomerase, the ends of chromosomes shorten
so they die rip

32
Q

what phase does dna replication occur

A

S phase

33
Q

how do we syncrhonise cells during experimentation

A

use chemicals to bring them all to beginning of S phase

34
Q

how do we visiualsie and detect DNA replication sites

A

modify thymidine nucleotides with Br
then can detect using antibodies that are fluorecently labelled
and antibodies attach to the Br nucleotides

35
Q

how many patterns of DNA replication can be observed?

A

5 patterns
they relate to the types of DNA being replicated:
-Euchromatin first
-Heterochromatin next

can be observed during S phase - see onenote for diagram

Molecular Biology BIOL10221 (17 decks)

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