DNA Replication Flashcards
(19 cards)
10 Requirements of DNA Replication
- DNA Polymerase
- Template DNA strand
- Primer with a free recessed 3’-OH
- All four dNTPs and Mg2+
- Origin of replication
- Helicase
- DNA gyrase
- Single-strand DNA binding proteins
- Primase
- DNA ligase
Overview of DNA Replication Properties
Enzyme - DNA Polymerase
Template - Single stranded DNA
Biochemical Mechanism of Action - Nucleophilic attack of the 3’-OH of the growing DNA chain on the alpha-phosphoryl of an incoming triphosphate
Driving Force - The subsequent hydrolysis of the eliminated PPi
How does the polymerase know which nucleotide to add next?
Incoming nucleotides are selected by their ability to form Watson-Crick base pairs
Main factors contributing of the accuracy of DNA replication
- Balanced nucleotide levels
- The polymerase mechanism
- Polymerase proofreading mechanisms
- The activity of DNA repair enzymes
Origins of Replication
- Specific locations in the DNA where replication machinery assembles and DNA replication begins
- Bacteria have a single origin of replication
- Eukaryotes have multiple origins of replication
What is the model of DNA replication?
Semi-discontinuous - 2 parent strands are synthesised in different ways
Leading strand - synthesised continuously, extending in the 5’ to 3’ direction of the advancing replication fork
Lagging strand - synthesised discontinuously in 5’ to 3’ direction as Okazaki fragments, as the parental DNA becomes newly exposed at the replication fork. Okazaki fragments are later joined together by DNA ligase.
DNA synthesis requires RNA primers
- DNA polymerases requires a free 3’-OH group to extend a DNA chain
- 5’ ends of Okazaki fragments contained segments of RNA complementary to the template DNA
- In E.coli RNA primers are synthesised by primase
- One primer required to synthesise leading strand
- Multiple primers required for lagging strand
- Mature DNA doesn’t contain any RNA because the RNA primers are eventually replaced with DNA
DNA Polymerases - Enzyme Activity of each polymerase
Pol I - Polymerisation 5’–>3’, Exonuclease 3’–>5’, Exonuclease 5’–>3’
Pol II - Polymerisation 5’–>3’, Exonuclease 3’–>5’
Pol III - Polymerisation 5’–>3’, Exonuclease 3’–>5’
DNA Polymerases - Enzyme Activity functions
Polymerisation 5’ → 3’ ADDS nucleotides in 5’ to 3’ direction
Exonuclease 3’ → 5’ REMOVES nucleotides in 3’ to 5’ direction - proofreading mechanism
Exonuclease 5’ → 3’ REMOVES nucleotides in 5’ to 3’ direction - excises nucleotides from single-stranded nicks, removal of RNA primers
Proteins Required for DNA Replication
Helicase - begins unwinding of DNA double helix
DNA gyrase - assists unwinding of DNA double helix
Single Strand Binding (SSB) Proteins - stabilise single strands of DNA
Primase - Synthesis of RNA primer
DNA Pol III - elongation of DNA strand by DNA synthesis
DNA Pol I - Removal of RNA primer and filling in gap with DNA
DNA ligase - closes last phosphoester gap to from phosphodiester bond
Helicase and DNA Gyrase
Helicase is a hexameric complex which separates DNA strands. Helicases translocate along a DNA strand, breaking H-bonds as they go and mechanically unwinding the helix in their path. This process is driven by the free energy of ATP hydrolysis. Unwinding of DNA is further facilitated by DNA gyrase.
Single-Strand Binding Protein (SSB)
SSB coats the newly separated DNA strands behind an advancing helicase so that they don’t reanneal to from dsDNA. The SSB coat also prevents ssDNA from forming secondary structures and protects it from nucleases. SSB must be stripped away before the ssDNA can be replicated by DNA pol.
Primosome
In E. coli, primer synthesis is mediated by the primosomes. This includes the helicase and an RNA-synthesising primase. Primase catalyses the polymerisation of an RNA segment of ~5-15 nucleotides in length. This primer provides the 3’-OH group for DNA elongation. The primosome is propelled towards the opening replication fork (5’–>3’). It reverses direction momentarily to synthesise complementary RNA primers for each Okazaki fragment in the lagging strand.
The Replisome
- In E. coli, a single multi-protein particle called the replisome catalyses the synthesis of both leading and laggin strands
- In contains two DNA Polymerase III enzymes, one to synthesise each new DNA strand
- A special “looping” arrangement of the lagging strand allows the replisome to move as a single unit in the direction of the advancing replication fork
- A continuously replicated leading strand and a series of RNA-primed Okazaki fragments separated by single-strand nicks is the result
DNA Pol III
The DNA Polymerase III “holoenzyme” is the complete, fully functional form of the enzyme
This includes the following components:
- Core Enzyme (with 5’ → 3’ polymerase and 3’ → 5’ exonuclease catalytic activities)
- Clamp Loader (multiple functions; mediates switch from primer synthesis to DNA synthesis)
- Sliding Clamp (functions as a sliding clamp to hold the holoenzyme complex to DNA, making the enzyme very processive)
DNA Pol III - Sliding Clamp
- The Pol III holoenzyme has a processivity of > 5000 residues due to the presence of its beta subunit
- The beta subunit forms a ring around the DNA that functions as a sliding clamp that can move along it, thereby keeping the Pol III holoenzyme from dissociating from the template
- Without this subunit interaction, the Pol III core enzyme would probably dissociate after replicating only ~ 12 residues
DNA Pol I
- Another E. coli enzyme that catalyses the synthesis of DNA
- Three different enzymatic activities of DNA Pol I occupy three separate active sites
Removal of RNA Primers by DNA Polymerase I:
- During replication, RNA primers are removed and replaced with DNA through the DNA Polymerase I - catalysed “Nick Translation” process
- Nicks are sealed by the action of the enzyme DNA ligase
- Note that this reaction requires energy in the form of ATP or NAD+ hydrolysis
Termination of Replication
- In E. coli, there is a replication terminus region flanked by ten “terminator sites”
- They acts as one-way valves, guaranteeing that the two replication forks will meet in the replication terminus region, even if one arrives their first
- The arrest of replication fork motion at Ter sites requires binding of Tus protein (Terminator Utilisation Substance)
Eukaryotic DNA Replication
- Eukaryotic and bacterial DNA replication mechanisms are remarkably similar, but the eukaryotic system is vastly more complex in terms of the amount of DNA to be replicated and the number of proteins required
- Several different modes of DNA replication occur in eukaryotic cells (which contain nuclear DNA in addition to mitochondrial DNA and, in plants, chloroplast DNA)
- Eukaryotes use several different DNA polymerases, each with different properties
- Unlike bacteria, eukaryotic DNA is replicated from multiple origins
