DNA Replication Machinery II Flashcards
1
Q
Opening of DNA double helix
A
- stable under normal conditions
- must be opened ahead of replication fork by helicase enzyme
- incoming deoxyribonucleoside triphosphates can form bps with the template strand
2
Q
Role of protein binding in DNA replication
A
- initiator proteins (PRO and viruses)
- origin recognition complex (EU)
- involved in the binding and recruitment of the helicase and other replication factors to the origin region to produce the pre-replicative complex
3
Q
PRO initiator protein
A
causes initial melting of double helix to expose small region of ssDNA
4
Q
EU origin of replication (ORC) proteins
A
do not melt dsDNA. Opening is achieved by helicase enzyme
5
Q
Role of DNA helicases
A
- separate the strands of DNA duplex and provide replication machinery with access to ssDNA templates
6
Q
Role of ssDNA binding proteins
A
- assist in the helix-opening processes
- protect and stabilise the exposed ss templates produced by helicase unwinding
7
Q
How do PRO initiator and EU ORC differ
A
- both involved in assembly of helicase
- differ in structure
- differ in loading onto origin dsDNA
- differ in ability to melt dsDNA
8
Q
PRO origin recognition proteins
A
- initiator protein DnaA is complexed with ATP and causes the initial melting of the double helix to expose a small region of ssDNA
- occurs before loading and activation of helicase
- controlled accumulation of DnaA at the origin ensures DNA replication only once per cycle
9
Q
Pathway of PRO initiator DnaA in E.coli
A
- DnaA in complex with ATP binds to methylated oriC minimal origin region
- several monomers form a filament around which oriC DNA is wrapped
- DnaA acts to melt the DNA strands
- DnaA filament contains ATPase domains which binds ssDNA, activating DnaA
- active DnaA compacts and forms open bubble complex
- 2 DnaB helicase hexamers are loaded in opposite directions
10
Q
EU origin recognition proteins
A
- ORC binds to and encricle dsDNA at origin
- ORC does not melt the dsDNA
- ORC complex is involved in loading the helicase in an inactive G1-phase of the cell cycle
- ATP binding required
- controlled assembly and activation of helicase at origin ensures DNA replication only once per cycle
11
Q
What is ORC
A
- complex of 6 proteins
- essential for initiation of DNA replication
12
Q
Structure of PRO DNA helicase
A
- ring-shaped
- hexameric
13
Q
Structure of EU DNA helicase
A
- ring-shaped
- hexameric
- includes complex of MCM proteins (posesses weak helicase activity, increases upon association)
14
Q
How to detect DNA helicases in vitro
A
- radioactively labelled DNA is incubated with the putative helicase protein in the presence of ATP and Mg ions
- if helicase is present, DNA will become unwound at sites of helicase binding
- partially unwound DNA will contain regions of ssDNA at these sites
- enzyme S1 nuclease is added, which digests ssDNA
- radioactively labelled dsDNA fragments left behind can be resolved by gel electrophoresis and detected by autoradiography
15
Q
How to detect DNA helicases using a fragment displacement assay
A
- piece of radioactively labelled DNA is hyrbidised to ss circular DNA containing complementary sequence
- substrate is incubated with putative helicase protein in presence of ATP and Mg ions
- if helicase is present, DNA becomes unwound and labelled fragment dissociates from circular molecule
- fragments can be resolved by gel electrophoresis and detected by autoradiography