DNA Replication – Process
Lecture 17
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Forms of DNA Helices
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DNA Replication
Template DNA (parent DNA)
Replication of parent DNA can theoretically generate 2 possible conformations of daughter
DNA
S Phase S Phase
M PhaseM Phase
Forms of DNA Helices
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DNA ReplicationDesign an experiment to differentiate these two
possibilities
DNA is unique among biomolecules due to very high concentration of Phosphorus
𝑃→ 𝑆❑32 +𝛾❑
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Grow cells in P32 enhanced
media
32P labeled DNA
Single cell cycle in unenhanced
media
Forms of DNA Helices
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DNA Replication
How do bacteria go about replicating DNA?
Linearize DNA for replication?
Keep DNA circular?
The experiment:Grow cells in 3H-thymidine
enhanced media
Forms of DNA Helices
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DNA Replication
Where does replication start and is it uni or bidirectional?
Heavy staining on both sides of the replication eye
indicates that replication is almost always bidirectional
in bacteria.
Bacterial replication also starts at the same spot indicating a single origin or replication
Forms of DNA Helices
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Dealing with DNA Superstructure
What enzyme is capable of modifying
DNA topology?Topoisomerase (DNA
Gyrase)
Parent DNA must be unwound at the replication fork
For this to occur at biological rates (1000 nt per second for E. coli), genomic DNA must twist at a rate of 100 rps.
E. coli’s genome is circular, so this is not possible!
Forms of DNA Helices
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Semi-discontinuous Replication
DNA’s 2 strands are replicated at the same time
DNA Polymerase synthesize DNA in the
5’ 3’ direction
So how does the other strand elongate?
5’5’ 3’
The ‘Lagging Strand’ is synthesized as small fragments (1000 – 2000 nuclotides in bacteria or 100-200 nt in eukaryotes) called Okazaki fragments.
Okazaki fragments are combined by a DNA Ligase
Forms of DNA Helices
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DNA Replication
How will a dNTP be added to the existing chain?
How might the reaction be activated?
Forms of DNA Helices
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Requirement for priming
This model of DNA replication requires a 3’ OH for strand elongation
Nucleophile
3’ 5’
5’
Analysis of Okazaki fragments indicated that the 5’ end was always composed of RNA
160 nucleotides in length
These will have to be replaced at some point
Forms of DNA Helices
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Enzyme Requirement for Replication
• DNA Polymerase• Catalyze the DNA chain elongation using the ‘parent’ strand as a template
• RNA Primer Synthesis• RNA Polymerase
• 460 kDa (E. coli)• catalyzes primer synthesis for leading strand only
• Primase• 60 kDa• Responsible for priming Okazaki fragment• Works synergistically with RNA Polymerase to prime leading strand
• Topoisomerase (Gyrase)• Unwind supercoiled template DNA• Energy dependent process
• SSB• Binds to ssDNA and prevents reannealing
• Ligase• Join Okazaki fragments
Forms of DNA Helices
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Formation of the Replication Fork
1. 4 DnaA proteins bind to the oriC region (recognizes 9 nucleotide segments)1. Additional DnaA monomers bind forming a histone like complex of tightly wound
DNA2. Localized melting of a 13bp repeats
2. DnaB (6 subunit helicase) binds and unwinds the DNA1. Topoisomerase functions downstream to relieve stress
3. Single Strand DNA binding proteins prevent the ssDNA from reannealing
Forms of DNA Helices
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Formation of the Replication Fork
DnaA helical filament – 8 monomers per turn
178 Å
• dsDNA wraps around the filament
• This increase Writhing number? • Enables some untwisting?
L = W + t
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The Big Picture (in E.coli)Topoisomerase: relieves
topological strain Helicase - unwinds dsDNA at replication fork
Primase: synthesizes RNA primers for lagging strand
DNA Polymerase III : elongate primed DNA from 5’3’
3’5’ exonuclease activity limits errors
Pol III elongates lagging strand until strained. It then releases the template and rebinds at a new primed location.
SSB: keeps ssDNA from reannealing
Pol I: closes any gaps in lagging strand and
replaces RNA primer
Ligase: seals off backbone nicks
Forms of DNA Helices
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DNA Polymerase
What do we need in a DNA polymerase?
• Template DNA binding site• Single Strand or Double Strand?
• Room for growing dsDNA
• dNTP binding site
• Mechanism to differentiate between the 4 possible dNTPs
• Self Correcting?
Forms of DNA HelicesE. coli DNA Polymerase I
Palm Domain • ~22 Å x 30 Å • Ideal shape to bind B-DNA • Lined with basic amino
acids
Thumb• Guides newly
formed DNA• Responsible for
processivity
Pol I• 3’ 5’ and 5’ 3’ exonuclease activity• Small N-terminal domain contains the 5’3’ exonuclease activity
• Completely independent from active site or 3’ 5 site
Fingers• Contains dNTP
binding site • Close in on
dNTP/Growing Strand once successful Watson-Crick base pairing made
Forms of DNA Helices
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Taq DNA Polymerase
Projected path of ssDNA template
5’
Thumb folds down over elongating dsDNA preventing it from escaping
Initial inspection of the groove between the thumb and fingers suggests the DNA would follow this cleft. This is NOT the case!Active Site
Growing Strand
Template
3 base pair closest to active site are A-Form
DNA
Projected path of ssDNA template
5’
Active Site
Growing Strand
Template
Forms of DNA HelicesTaq DNA Polymerase
Thumb folds down over elongating dsDNA preventing it from escaping
Initial inspection of the groove between the thumb and fingers suggests the DNA would follow this cleft. This is NOT the case!
Forms of DNA Helices
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Taq DNA Polymerase
Projected path of ssDNA template
dNTP Binding Site – allows for rapid sampling of the dNTPs
Thumb
Knuckles?
Forms of DNA Helices
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Taq DNA Polymerase
Growing Strand
Template
Watson-Crick Base Pair between Template and incoming dNTP at active
Mg situated for activation
of 3’OH
DNA polymerases apparently select incoming dNTP based on SHAPE of Watson-Crick base pair (Purine-Pyrimidine)
5’ end of growing strand is 2’-3’-dideoxy
CTP
Only one combination will provide most favorable pair
Forms of DNA Helices
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3’ 5’ Exonuclease
The structure of E. coli Pol I has been solved with DNA arranged in the 3’5’ exonuclease active site
Growing strand peels away from active site
The base pair closest to the polymerization active site is significantly weakened
Exonuclease site (Zn2+ activated mechanism)
Which bond will be cleaved?
NEED 3’-hydroxyl for elongation
Forms of DNA Helices
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Model for Proofreading in DNA Polymerases
• dNTPs are in rapid equilibrium at active site
• When reaction occurs, proper base pair will form a strong tight bonding pair
• When an error occurs, the base pair is not as tight, resulting in increased favorability for the growing chain to be positioned at the 3’5’ exonuclease site
• A-form DNA of DNA @ polymerization active site makes this process easier (less tightly wrapped and less stable helix)
Forms of DNA Helices
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5’ 3’ Exonuclease
Pol I from Thermus aquiticus lacks 3’5’ exonuclease activity
N-terminal domain contains 5’ 3’
exonuclease
Taq polymerase can translate a single nick in DNA by a mechanism that involves both the polymerase
active site and the 5’3’ exonuclease active site
Forms of DNA Helices
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Taq DNA Polymerase ExonucleaseWhat process in DNA replication requires a
5’ 3’ exonuclease process?
Need to remove RNA primers from lagging strand!
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DNA Ligase
• Needed to seal the phosphodiester backbone following removal of the RNA primer by Pol I
• Energy Dependent reaction
• Hydrolysis of ATP or NADH
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DNA LigasepdbID 2OWO
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DNA Ligase
• AMP covalently bound to active site
pdbID 2OWO
AMP phospoamide intermediate with Lysine shown
biochemically
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DNA Ligase Reaction
AMP phospoamide intermediate forms with Lys
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DNA Ligase
Covalent Lys-AMP intermediate displaced by 5’ Phosphate of nicked DNA
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DNA Ligase3’ OH attacks 5’ Phosphate displacing the AMP
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DNA Helicase
• Helicase functions as a hexamer
• Each subunit has a babbb fold
• Forms a clamp around the DNA that slides and unwinds dsDNA
• Unwinds dsDNA by ‘active rolling’ mechanism
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DNA Primase
• Primase must be able to oppose the direction of translocation
• Forms a non-covalent bond with DnaB (helicase)
• Synthesizes primers ~11 nt in vivo but ~60 in vitro
• Basic groove nonspecifically binds to ssDNA and directs it to the active site
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Single Stranded DNA Binding Protein
• SSB binding to DNA prevents: • reannealing• Stem loop structures (self
complimentary sequences)
• Nuclease degradation
• Very basic proteins• Bind to ssDNA with no sequence
specificity• Binds as a tetramer• Can bind in a number of different
conformations
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Replication Termination
There are multiple termination elements in E. coli’s genome.
Each are similar sequences long and promote binding of TUS proteins
Consensus Sequence:ANNTAGTATGTTGTAACTA
Clockwise moving replication forks pass through TerE, TerD and TerA but stop at TerC
CCW moving replication forks pass through TerG, TerF, TerB and TerC but stop at TerA
This verifies that each replication fork will finish at same place in genome
Makes specific contacts with
DnaB – termination is dependent on this interaction