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Gene Regulation and Expression > DNA Replication Machinery II > Flashcards

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
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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
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3
Q

PRO initiator protein

A

causes initial melting of double helix to expose small region of ssDNA

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4
Q

EU origin of replication (ORC) proteins

A

do not melt dsDNA. Opening is achieved by helicase enzyme

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5
Q

Role of DNA helicases

A
  • separate the strands of DNA duplex and provide replication machinery with access to ssDNA templates
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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
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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
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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
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9
Q

Pathway of PRO initiator DnaA in E.coli

A
  1. DnaA in complex with ATP binds to methylated oriC minimal origin region
  2. several monomers form a filament around which oriC DNA is wrapped
  3. DnaA acts to melt the DNA strands
  4. DnaA filament contains ATPase domains which binds ssDNA, activating DnaA
  5. active DnaA compacts and forms open bubble complex
  6. 2 DnaB helicase hexamers are loaded in opposite directions
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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
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11
Q

What is ORC

A
  • complex of 6 proteins
  • essential for initiation of DNA replication
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12
Q

Structure of PRO DNA helicase

A
  • ring-shaped
  • hexameric
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13
Q

Structure of EU DNA helicase

A
  • ring-shaped
  • hexameric
  • includes complex of MCM proteins (posesses weak helicase activity, increases upon association)
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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
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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
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16
Q

What do the control lanes indicate in a fragment displacement assay measuring DNA helicase activity

A
  • relative positions of the bands for the partial duplex untreated with the test protein and the labelled fragment dissociated from the ss circular DNA by boiling
  • presence of a labelled band which does not correspond to the initial DNA partial duplex substrate, but which corresponds to the dissociated labelled fragment, suggests that the test protein contained helicase activity
17
Q

Loading of the PRO E.Coli DnaB helicase

A
  • opening of hexameric ring helicase DnaB occurs upon binding of helicase loader DnaC
  • the ssDNA produced by origin melting (by ip DnaA) enters through crack in ring and binds the central channel of DnaBC complex
  • DnaB ring closure and DnaC release leave the active DnaB helicase loaded on the ssDNA
18
Q

Loading of EU MCM complex helicase

A
  • hexamer of inactive MCM helicase complex is loaded onto duplex DNA by combined actions of ORC and specialised loader proteins Cdc6 and Cdt1
  • open ring of ORC subunits 2-5 binds Cdc6 and this hexamer encircles the origin duplex DNA
  • combined actions of ORC-Cdc6 and coloader Cdt1 load an open-ring hexameric MCM complex onto duplex DNA
  • Cdc6 and Cdt1 are released upon binding of MCM helicase complexes
19
Q

Replicative DNA helicases in PRO and EU

A

in both PRO and EU, two helicase hexamers become loaded onto complementary DNA strands in opposite directions at the origin of DNA replication

20
Q

Single-stranded DNA binding proteins

A
  • binds to ssDNA cooperatively without covering bases and are displaced from ssDNA as DNA Polymerase creates new complementary strand
  • assist in DNA helix-opening processes by stabilising the unwound ss conformation and protecting ssDNA from hydrolysis/formation of hairpin helices
  • facilitate transfer of 3’OH end of newly synthesised RNA (primer)/DNA between polymerases
21
Q

DNA Polymerase action creating new strand following strand separation

A

DNA polymerase cannot begin DNA chain synthesis de novo, there is a need for a priming event to provide the RNA with the essential 3’OH group from which DNA Polymerase can initiate DNA synthesis

22
Q

What priming mechanisms can take place to provide the 3’-OH group

A
  • nicking of DNA duplex
  • presentation to the DNA polymerase of the 3’OH group of a protein-bound nucleotide
  • use of the 3’OH of a pre-formed RNA bound near the origin
23
Q

Priming events in PRO and EU systems

A
  • involves a primase
  • primase synthesises and RNA primer in open region near origin
  • primer 3’OH end must be transferred to DNA polymerased
  • DNA polymerase can synthesise DNA
  • leading strand only requires one priming event at each origin
  • lagging strand requires a series of priming events for each Okazaki fragment
24
Q

Mechanism of action of primases

A
  • bind ssDNA templates
  • to enable frequent binding on lagging strand, most primases synthesise RNA complementary to almost any ssDNA they are bound to
  • RNA synthesis by primase occurs at low fidelity
  • following leading and lagging strand DNA synthesis, RNA primer is removed and replaced with DNA (high fidelity)
25
Q

PRO E.coli primase synthesis

A
  • DnaG
  • transiently associated with DNA polymerase
  • Zn binding region; protein-protein/protein-DNA interactions
  • catalytic domain; polyribonucleotide synthesis
  • variable C-terminal region (helicase/binding domain for a helicase)
26
Q

EU primase synthesis

A
  • within a complex of 4 proteins, includes DNA polymerase (alpha)
  • on both leading and lagging strands, following synthesis of RNA primer by primase, DNAPalpha covalently attaches 1st deoxyribonucleotide to the free 3’OH end
  • elongates DNA chain to produce initiator

Gene Regulation and Expression (10 decks)

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