DNA Replication Flashcards
Which bacteria was used to make a fundamental aspect of our knowledge of the natural world
E. coli
What are the 3 proposed models of replication
Conservative, semiconservative, dispersive
Describe the Meselson/Stahl experiment
Cells were grown in media with heavy isotope of nitrogen.
DNA is extracted from these cells
The cells are shifted to media with normal nitrogen.
DNA is extracted from these cells after another generation
DNAs that have 2 strands with N15(heavy) will be heavy and band at a low position in a centrifugation gradient
DNAs that have 2 strands with N14 will be light and migrate high up in the same gradient
DNAs with one strand N15 and one N14 will be intermediate and be between the heavy and light duplexes
showed it was semi-conservative
Where does the replication of bacterial start and how does it proceed
starts at origin of replication and moves bidirectionally
what is the structure called where the parental strands are separated and the new DNA synthesis occurs
replication fork
Do eukaryotic and prokaryotic chromosomes have one or multiple origins of replication
Eukaryotic: multiple
Prokaryotic: single
What does DNA polymerase do
enzymes that catalyzes template-dependent synthesis of DNA from its deoxyribonucleotide 5′-triphosphate precursors– the linking together of dNTPs into a long chain is called DNA polymerization
Which direction do DNA strands grow
5’-3’
5 properties of DNA polymerases
- Transfers phosphate from dNTP to the last added deoxynucleotide, the last two phosphates are released as pyrophosphate - repeated many times
- Require a primed template strand to direct the synthesis
- can only move in the 3’-5’ direction of the template, polymerizes the new strand in the 5’-3’ direction
- the product is a new dsDNA with strands arranged in antiparallel orientation. one strand is the old template strand and one strand is the newly synthesized strand
- Can only extend pre-existing DNA or RNA chains (unlike RNA polymerases they cannot create new chains de novo)
What direction is the leading and lagging strand synthesized
leading strand (continuous): 5’-3’
Lagging (discontinuous): 3’-5’
What are the 2 DNA polymerases involved in DNA replication? describe them
DNA Polymerase III: Large complex composed of the main replicative polymerase that extends the growing DNA chain and many accessory factors that act to make new DNA at the replication fork
DNA Polymerase I: Polymerase that plays a special role in lagging strand replication and DNA repair
What are nucleases
Enzymes that degrade DNA (or RNA) by breaking the phosphodiester bonds
What are the two types of nucleases
Endonucleases: break the DNA in the middle of a DNA strand
Exonucleases: remove nucleotides only from an end
2 classes of exonucleases
Need 3 classes because of different polarities of DNA strands
1. 5’ exonucleases digest DNA strands from the 5’ end and move 5’ to 3’
2. 3’ exonucleases digest DNA strands from the 3’ end and move 3’ to 5’
3
example of an essential exonuclease activity
One of the domains of DNA pol I involved in proofreading
Describe the process of the proofreading 3’-exonuclease
- the 3’ terminus repositions back to the polymerase site
- polymerase incorporates the correct nucleotide
T/F the proofreading site is distinct from the polymerization site
true
3 major domains of nick translation by Pol I
DNA polymerase
3’-5’ proofreading exonuclease
5’-3’ exonuclease
describe Nick translation by Pol I’s 5’- exonuclease
At the nick(gap between lagging strand fragments), Pol I degrades the RNA primer in the 5’-3’ direction, Releasing NMPs, and simultaneous extending the 3’ terminus with dNTPs in the same direction. The net result is movement of the nick in the 5’-3’ direction along the DNA until all RNA is removed. DNA ligase then seals fragments
Degrades RNA primer and as the primer is degraded, it extends the 3’ terminus with dNTPs in the same direction.
How many proteins are associated with DNA pol III? Together they are known as _____
9
DNA pol III holoenzyme
DNA pol III holoenzyme composition
3 pol III cores
3 Beta clamps
1 clamp loader
what are the 3 Pol III cores? What are their functions?
Alpha = polymerase
epsilon = proofreading
beta = binds epsilon
look at picture
3 Beta clamp sub units
Clamp loader subunits
Tau, delta, and delta prime
f
What are the 5 important parts of the DNA pol III holoenzyme
alpha, epsilom, beta, delta and delta prime, tau
1) the a subunit is the polymerase
2) the e subunit is the 3’ exonuclease for proofreading
3) the b subunit is the sliding clamp
4) the d and d’ subunits are the clamp loader
5) the t subunit holds the complex together so that the polymerases that act on the two strands are held together
What is the beta clamp for?
DNA polymerase must be tethered to the DNA via a sliding clamp
It encircles the DNA as a ring and keeps the DNA polymerase on the template DNA allowing synthesis of very long chains without stopping
Beta clamp increases the speed and processivity of DNA polymerization
Why does the Beta clamp increase pol III processivity
Converts pol III from distributive to processive.
When Pol III detaches from the DNA, it stays attached to the beta clamp and rapidly reattaches to the DNA
What does the clamp loader do
Because the sliding clamp is a closed doughnut that encircles the DNA it needs a special complex that opens/recloses the sliding clamp ring to load the sliding clamp and to allow its removal from the DNA when its job is completed
The gap of the clamp loader is between which two sub units
Delta’ and delta
describe the mechanism of the clamp loader
- ATP binds to gamma subunit and opens clamp
- Beta clamp binds primed DNA
- ATP hydrolysis ejects clamp loader allowing clamp to close around DNA
What is the Replicative DNA helicase
Unwinds DNA
Called DnaB
unwinds the parental duplex DNA into 2 template strands
Why does DnaB associate with pol III
Unwinding alone is slow (35 bp/s) but when it associates with DNA pol III it unwinds DNA much faster (700 bp/s)
Unwinding is the rate-limiting step of bacterial DNA replication
What is the rate-limiting step of bacterial DNA replication
DnaB
What does Topoisomerase do
DNA unwinding by the replicative DNA helicase, DnaB, causes overtwisting ahead of the replication fork that would cause replication to stop if it was not untwisted by the action of a topoisomerase called DNA gyrase
What is the topoisomerase in bacteria called
DNA gyrase
What do primases do? what does it need to be active?
Enzymes that make short RNA primers to initiate synthesis of new DNA strands
Required for DNA replication because DNA polymerases cannot start the synthesis of a new DNA strand
Must bind to DnaB to be active
The RNA to which DNA polymerase adds deoxy-nucleotides is known as a primer
What is DNA ligase
Form phosphodiester bonds between the ends of DNA chains to form longer chains
During replication DNA ligase joins the 5’phosphate group to the 3’ OH at the end of another DNA joining them
seals Okazaki fragments after removal of RNA primers
What does single-stranded DNA binding protein (SSB) do
protects the DNA unwound by the replicative helicase from nucleases and other damage and prevents the ssDNA from reannealing or forming secondary structures that would impede progression of the replication fork
What is the trombone model of the replication fork
- Lagging-strand loop formation
- Loop growth
- Loop disassembly
and clamp loading
Summary:
A description of DNA
replication on the lagging strand, with its repeated cycles of loop growth and disassembly, by analogy with the movement of a slide on a trombone
Full:
The trombone model of replication fork function. The lagging-strand polymerase extends the 3′ terminus of an Okazaki fragment in the opposite direction to fork movement, yet this polymerase is part of the replisome and thus moves with the fork. The opposed directions result in formation of a DNA loop for each Okazaki fragment. As multiple Okazaki fragments are synthesized, loops repeatedly grow and are released, similar to the movement of a trombone slide as the instrument is played
Why is DNA replication semi-discontinuous
Synthesis of leading strand is continuous
Synthesis of lagging strand is discontinuous
What are Okazaki fragments
Lagging strand is synthesized in fragments of about 2000 nucleotides (Okazaki fragments)
How many primers are needed for the leading and lagging strand
The primase (DnaG) synthesizes these primers for the lagging strand
primase makes primers for lagging and leading strand synthesis at oriC (once for the leading strand and many times for the lagging strand)
How are primers removed from Okazaki fragments
RNA primers are removed by the action of DNA pol I
DNA pol I replaces the RNA primer with DNA in a process called nick translation that employs the 5’-exonuclesae and DNA polymerase subunits working in tandem
What are the steps in Okazaki fragment processing
1) Pol III core dissociates from Beta clamp after completing Okazaki fragment, and Pol I targets the Beta clamp to remove the RNA primer
2) Dissociates after replacing the primer with DNA, and DNA ligase targets the Beta clamp to join the Okazaki fragments
Why are eukaryotic replication forks more complex than bacterial
More proteins are needed to achieve the various needed functions
Eukaryotes contain all the proteins that function in a bacterial replisome, but most components have more subunits than the bacterial proteins, and several additional proteins function at the eukaryotic replication fork.
Describe the PCNA clam and the RFC clamp loader
The PCNA (Proliferating Cell Nuclear Antigen) clamp is composed of a heterotrimer that assembles into a donut shaped ring that looks almost identical to the b clamp
The RFC (Replication Factor C) clamp loader has 5 subnunits that assembles PCNA onto primer template junctions in a similar manner to the bacterial clamp loader
Describe the eukaryotic primase
4-subunit complex called DNA polymerase alpha
Pol a has primase activity that lays down a short RNA primer but also has a DNA polymerase subunit that extends the primer with about 25-40 nt of DNA
This extended primer is then used by a replicative DNA polymerase for full extension
Which DNA polymerases synthesize the leading and lagging strands in eukaryotes
DNA polymerase d (delta)
DNA polymerase e (Pol e, epsilon)
What do Pol delta and epsilon bind to promote processive DNA polymerization
Bind PCNA
What are the essential proteins n the replication progression complex
Ctf4 and Mcm10 are essential
What is the SSB in eukaryotes
RPA (replication protein A)
Why is eukaryotic replication slow
Eukaryotic replication is much slower than in bacteria with a synthesis rate of ~30-50 nt/s compared to 700 nt/s for bacteria
Okazaki fragments are much shorter in eukaryotes 100-200nt compared to 1000-2000
Packaging into chromatin slows down DNA replication rate
What is the MCM complex
a ring-shaped eukaryotic helicase complex that acts at the replication fork. It is composed of six homologous, but nonidentical, ATPase proteins and interacts with two other proteins to form the CMG complex.
What is the CMG complex
DNA helicase in eukaryotes
a complex of the proteins Cdc24, MCM helicase, and GINS proposed to function in the eukaryotic replisome
DNA polymerase alpha, delta, and epsilon
DNA polymerase α (Pol α): a eukaryotic DNA polymerase with both primase and error-prone DNA polymerase activities. The enzyme synthesizes an RNA primer on a DNA template and then extends it with DNA
DNA polymerase d (Pol d): a eukaryotic chromosomal replicase with both DNA polymerase and 3′→5′ exonuclease activities. It acts on the lagging strand of the replication fork.
DNA polymerase ε (Pol ε): a eukaryotic chromosomal replicase with both DNA polymerase and 3′→5′ exonuclease activities. It acts on the leading strand of the replication fork
What is the replisome progression complex
a large protein assemblage that is part of the eukaryotic replication machinery; includes proteins that move with the replication fork, proteins that participate in affixing Pol α in the replisome, and several nonessential proteins thought to control the rate of replication during times of cellular stress (includes Mcm10 and Ctf4)
