L17-18 DNA Replication Flashcards
Meselson & Stahl (1958)
- DNA is synthesised in a heavy medium (15 N)
- DNA is then moved to the light medium (14 N) and replicated one cycle
- This is then repeated for 2, 3 cycles etc.
- Density of DNA is measured after each stage using density-gradient equilibrium sedimentation in CsCl
- This showed an intermediate weight DNA after replicating the the light medium once
- This supported semi-conservative replication
DNA polymerase was discovered by…
Korburg (1958)
DNA polymerase adds…
dNTP precursors to DNA
DNA polymerase is a template…
Directed enzyme
DNA polymerase ( does / does not ) require a primer
Does
The template strand catalyses the formation of phosphodiester linkages if…
The bases are complimentary
DNA polymerase catalyses…
Nucleophilic attack on the inner phosphorus of the new molecule by the 3’ hydroxyl of the previous DNA molecule
DNA polymerase moves from ( 3’ / 5’ ) to ( 3’ / 5’)
5’ to 3’ (relative to the new strand)
DNA polymerase can remove mismatched nucleotides as it has…
3’ to 5’ exonuclease activity
DNA replication in E.Coli has…
1) two origin points
2) three origin points
3) one origin point
4) many (>3) origin points
3) one origin point
Known as oriC
OriC contains 5 copies of…
dnaA binding sequence
The dnaA hexamer binds to the sequences at oriC, which causes (2)…
- Strain on the DNA backbone
* unwinding at the weak AT rich array upstream of OriC
dnaB helicase is recruited by…
dnaA
dnaB helicase is loaded around ( ssDNA / dsDNA ) at the ( 3’ / 5’ ) end
ssDNA at the 5’ end
ATPase-dependent translocation is carried out by…
dnaB helicase
The model of dnaB as loading around one strand of the DNA is known as the…
Strand exclusion model
Exposed single stranded DNA is stabilised by the binding of…
SSBs (single-stranded binding proteins)
SSBs are
a) monomeric
b) trimeric
c) tetrameric
d) hexameric
c) tetrameric
They are made up of four monomers
Trying to unwind a helix can cause…
Supercoiling of the remaining helix!
To relax the dsDNA and prevent supercoiling…
Topoisomerase I is recruited
Topoisomerase II prevents tangling and…
Catalyses the untangling of DNA duplexes by allowing two strands to pass through one another
Topoisomerase I relaxes the DNA by…
1 turn
DNA replication is:
a) initiated without the need for a primer
b) initiated by a DNA primer
c) initiated by an RNA primer
d) initiated by either a DNA or RNA primer
c) initiated by an RNA primer
This might be evidence for the RNA world hypothesis
Primase can:
a) initiate but not extend
b) extend but not initiate
c) initiate and extend
d) none of the above
a) initiate but not extend
Conversely, DNA polymerase can:
a) initiate but not extend
b) extend but not initiate
c) initiate and extend
d) neither extend nor initiate
b) extend but not initiate
Hence why a the primer is needed
Replication of the leading strand is catalysed by DNA polymerase ( I / II / III / IV )
DNA polymerase III
DNA polymerases I & II are involved with lagging strand synthesis
The DNA polymerase III core is made up of the (3)…
α, ε and θ subunits
alpha, epsilon and theta
The α-subunit of DNA polymerase III catalyses…
Base addition and maintains the correct geometry for successful base pairing!
The error rate of DNA polymerase III is reduced from 1 in 10^3 to around 1 in 10^6 by the…
Exonuclease which performs a proofreading function, cutting off incorrect base pairs and sending the strand back
The β2 subunit is the…
Sliding clamp
The β2 subunit increases the processivity of the enzyme, resulting in a rate of…
1000 nucleotides per second!
Unlike dnaB helicase, the hole in the similarly donut-shaped β2 subunit is…
a) larger (35Å in diameter) allowing dsDNA through
b) smaler (35Å in diameter) not allowing dsDNA through
c) larger (70Å in diameter) allowing ssDNA through
a) larger (35Å in diameter) allowing dsDNA through
The lagging strand requires…
a) a primer at the start
b) a primer for each fragment
c) a primer every 40-50 nucleotides
b) a primer for each fragment
A significant amount of newly synthesised DNA exists as small pieces, known as…
Okazaki fragments
DNA polymerase can only synthesise in the…
5’ to 3’ direction
DNA polymerase I…
removes RNA primers and synthesises DNA
DNA polymerase I is ( faster / slower / the same speed) as DNA polymerase III
Slower at about 10nt/s versus 1000nt/s
DNA ligase fills in the…
‘nicks’ between okazaki fragments, catalysing phosphodiester bond formation
DNA polymerase I has ( 5’ → 3’ / 3’ → 5’ ) exonuclease activity
5’ → 3’
The trombone model describes the… 🎺
Lengthening and shortening of the visible ssDNA loop of lagging strand created by DNA polymerase III holoenzyme during the replication process
There are an array of DNA repair proteins which are at work…
Constantly! 👷♀️
Dideoxy Sequencing is a method of DNA sequencing pioneered by…
Sanger (1977)
