Chapter 13 - DNA Replication Flashcards
Initiation
1) Parental strands are separated
2) The ssDNA are stabilized
3) A replication bubble is
Elongation
1) Assembly of replisome, a multiprotein structure, that undertakes DNA synthesis at the replication fork
2) Replisome moves along the DNA
3) Parental strands unwinds
4) Daughter strands are synthesized.
Termination
The joining of the replicons after which the duplicated
chromosomes are separated.
Conditional Lethal
Replication mutation. Example: Temperature sensitivity.
Because inability to replicate DNA is fatal, must generate mutants that are able to replicate only under permissive conditions but die otherwise.
Direction of DNA synthesis
5’-3’
Precursor for DNA synthesis
Nucleoside triphosphate that loses the terminal two phosphate groups in the reaction.
DNA synthesis is required for
Semi-conservative DNA replication and DNA-repair
Replicase
DNAP III. One bacterial DNA polymerase undertakes semiconservative replication
DNAP α
High-fidelity replicase in eukaryotes. Nuclear replication.
DNAP δ
High-fidelity replicase in eukaryotes. Lagging strand.
DNAP ε
High-fidelity replicase in eukaryotes. Leading strand.
DNAP γ
High-fidelity replicase in eukaryotes. Mitochrondrial replication.
E. coli DNA polymerase I
Unique 5’ and 3’ exonuclease activity.
A 103 kD polypeptide that can be cleaved into a large subunit (68 kD) and a small subunit (35 kD).
DNAP I Large Subunit
Klenow Fragment
- Polymerase
- 3’-5’ Exonuclease
DNAP I Small Subunit
5’-3’ Exonuclease
The fidelity of replication relies on
Specificity of base pairing.
Two classes of DNA polymerase-induced errors:
- Substitutions
2. Frameshifts
Correction of a substitution error
Proofreading scrutinizes newly formed base pairs.
Correction of a frameshift error
Reduced with greater enzyme processivity.
Processitivity
The tendency of the enzyme cycle on and off the DNA strand/template.
Fidelity mechanism for high-fidelity DNAP
The geometry of the base-pairing. Improper fitting stalls the DNAP. Moves the DNAP back to remove the mismatched base.
Three domains of many DNAP
Form a large cleft that resemble a right hand.
1. The “palm” includes a conserved sequence motifs that
constitute the active site.
2. The “fingers” (variable) involved in positioning the DNA.
3. The “thumb” (variable) binds DNA and important for processivity.
DNAP nuclease activity
Resides in a completely different domain from large cleft/hand that positions DNA.
What exposes the template to an incoming nucleotide while held in the DNAP “hand”
A sharp turn in the parental strand exposes the template to the incoming nucleotide.
The 3’end to which the nucleotide is being added is anchored by the fingers & palm.
Contacts between DNAP “hand” and DNA
Via phosphodiester bond
Rotation of the DNAP during DNA replication
The fingers rotate 60-degrees while the thumb rotates 8-degrees towards the palm.
Rotational changes is cyclic.
Is the lagging strand DNA replication continuous or discontinuous?
The lagging strand is synthesized in sequential fragments (Okazaki fragments) or discontinuously in the 5’-3’ direction and subsequently joined/ligated together.
Hence it is semidiscontinuous replication.
What is required to generate and maintain ssDNA for replication?
- A helicase to separate/unwind the strands of
DNA using energy provided by hydrolysis of ATP. - A single-stranded binding protein (SSB) is required to
maintain the separated strands.
What do all DNAP require ?
A 3’-OH priming end to initiate DNA synthesis or a primer.
Methods to provide the free 3’-OH DNAP require to initiate DNA synthesis?
- RNA primer
- a nick in DNA
- a priming protein
How many times is the RNA primer uses in leading versus lagging strand?
- One (1) time in Leading Strand
- Multiple times in Lagging Strand
Primase
Synthesizes the RNA chain that provides the priming end
What does priming of replication (starting DNA synthesis) on double stranded DNA always require?
A replicase (DNA Pol), helicase (DnaB), SSB, and primase (DnaG).
E. Coli priming reactions
- The oriC system - involves the DnaG primase at the fork.
- The φX system - requires a primosome to restart
replication at a damaged fork.
How many DNAP catalytic units are required to synthesize the leading and lagging strands?
Two dimeric DNAP catalytic units are required:
- One enzyme unit elongates the leading strand processively in the same direction.
- The other enzyme unit must move in the reverse direction to synthesize an Okazaki fragment backwards
Each monomeric unit of the E. Coli replicase (DNAP III) dimeric structure contains:
- A catalytic core that includes the α, ε, and θ subunits.
- A dimerization subunit or τ that links 2 catalytic cores.
- A processivity component that includes the β-ring clamp
(holds the catalytic core on the template) and γ complex
clamp loader.
Catalytic core includes
α, ε, and θ subunits
T-unit
Dimerization subunit - links 2 catalytic cores.
Processivity component
Includes the β-ring clamp (holds the catalytic core on the template) and γ complex clamp loader.
Clamp loader
Uses ATP hydrolysis to open the β-ring/bind β-subunits to a template.
Binding DNA changes the conformation of the β-subunits. Enhances the recruitment of the catalytic core.
Brings the enzyme core to the DNA.
Dimerization component binds the 2 core enzyme.
Each enzyme core synthesizes one of two new strands of DNA.
Clamp loader dissociates (but remains close) once the clamp is positioned on the DNA.
Cyclic process at the lagging strand.
How does the β-ring clamp associate with DNA?
The β-subunit of DNA Pol III holoenzyme is a head-to-tail dimer. Forms a ring completely around the DNA.
The β-ring clamp “skates” along the DNA. No bonds.
Associates with DNA via the intermediate water molecules
between the clamp and the DNA.
Helicase DnaB
Responsible for interacting with the primase DnaG to
initiate each Okazaki fragment.
Contacts the τ subunits of the clamp loader to connect the helicase, primase, and the catalytic core.
Trombone model
Synthesis of the leading strand creates a loop of ssDNA on the lagging strand. The enzyme complex on the lagging strand that “pulls” the ssDNA through the clamp while synthesizing the new strand.
Low-fidelity DNAP are also known as
Error-prone polymerases.
How many subunits do replicases have?
Four (4) subunits (tetramers) for catalysis, priming, processivity, and proofreading.
DnaB/MCM complex
Helicase
DnaC/Cdc6
Loading helicase/primase
SSB/RPA
Single-strand maintenance
DnaG/DNAPα/primase
Priming
τ
Holoenzyme dimerization
Pol I/FEN1
RNA removal
Ligase/Ligase 1
Ligation
Pol III core/Pol ε/Pol δ
Catalysis
Pol ε
Elongates the leading strand
Pol δ
Elongates the lagging strand
β/PCNA
Processivity
Three different DNAP make up eukaryotic replication fork
Pol α/primase (priming)
DnaB/MCM (helicase unwinding)
β/PCNA (processivity)
Which DNA polymerase expresses a nuclease activity (E. Coli or mammal)?
E. coli DNAP I express nuclease activity to remove a primer.
How do mammalian DNAP remove a primer?
- RNase H makes an endonucleolytic cleavage to
generate a “flap”. - FEN1 is a 5’-3’ exonuclease that recognizes and cleaves the RNA primer “flap”.
Two options when replication fork stalls at a damaged base or nick in DNA
Lesion bypass OR recombination.
Lesion bypass
Occurs during replication with an error-prone DNAP on a template that contains a damaged base.
Error-prone DNAP can incorporate a noncomplementary base into the daughter strand.
The mismatch is repaired at a later time.
Primosome
Required to restart a stalled replication fork after the DNA has been repaired.
Binds and functions to reload a new DnaB to the reinitiate
replication.
Recombination (after replication fork stalls)
The damaged sequence is excised. The complement of the other daughter duplex crosses over to repair the gap.
Allows replication to continue.
Termination of replication in E. coli
Called ter site.
~23 bps
Function is unidirectional.
E. coli have 2 cluster of 5 ter sites.
Each set allows the replication fork into the termination region, but does not allow it out the other side = “replication fork trap”.
The linear eukaryotic chromosomes most likely use a modified form of the ligation process.
