Lecture 9 - Enzymology of DNA replication Flashcards
Who established the directionality of DNA synthesis?
The Okazaki
What is the leading and lagging strand?
Leading: synthesised continuously - same direction as the replication fork
Lagging: synthesised discontinuously - 5’to4’ synthesis proceeds in the opposite direction
What are Okazaki fragments?
Small fragments of strand on the lagging strand only
Consequence of synthesis of new DNA in one direction only
Occur away from replication fork
Why is there no 3’to5’ synthesis of DNA?
One nucleotide removed leaving 5’ phosphate but a triphosphate is required
No high energy bond can be cleaved, no reaction can be processed
High energy bond required for incorporation of nucleotide
What is the life cycle of a M13 bacteriaphage?
Injection via the pilus
Single stranded DNA is replicated and becomes double stranded
Packed into fresh phage and secreted
What did Okazaki and Kornberg discovery with the beginning to DNA replication?
DNase cannot completely destroy Okazaki fragments
Primer for an Okazaki fragment is RNA, not DNA
Little pieces of RNA, 10-12 bases long were left
How are RNA primers synthesised?
DNA primase is a rifampicin-sensitive DNA directed RNA polymerase
Synthesizes an RNA primer to initiate DNA synthesis on lagging strand
RNA polymerases don’t require a primer
How is the lagging strand synthesised?
- New RNA primers are synthesised by DNA primase
- DNA Pol III extends RNA primer using dNTPs to make Okazaki fragments on lagging strand
- As replication fork separates fork separates more DNA, new primers are laid down by DNA primase
- Old primers erased by 5’to3’ exonuclease
- Gap sealed with DNA ligase, joining Okazaki fragment to growing chain
How is the Okazaki fragment joined by DNA ligase?
DNA ligase uses ATP, releasing pyrophosphate and attaching AMP to 5’ phosphate of downstream fragment
AMP released, phosphodiester bond formed between 3’ -OH of upstream Okazaki fragment and 5’ phosphate of downstream fragment
Sealing needs ATP
New bond seal gap
What is the clamp holder?
Hold 2 molecules of Pol III
Has helicase and DNA primase
What is leading strand synthesis?
- DNA helicase unwinds DNA helix, separating strands
- DNA primase synthesises DNA primer on leading strand template
- Primed duplex is captured by Pol III
- New clamp halves maintain in clamp holder
5.Clamp holder transfers 2 halves of B-clamp to Pol III
- Helicase continues to unwinds, and Pol III replicates the leading strand continuously
What is processivity?
Measure of an enzyme’s ability to catalyse consecutive reactions without releasing substrate
What is particular of Pol III?
Has low processivity
Can only make short stretches of DNA before it falls off DNA
What is the process of lagging strand synthesis?
- DNA primase produces RNA primer
- Primed duplex is clamped by Pol III forming loop
- Helicase continues to unwinds, Pol III extends new primer on lagging strand until Okazaki fragment has been pulled back to Pol II
- Lagging strand and template are unclamped
- DNA primase primes the lagging strand template
- DNA Pol I and DNA ligase repair the gap
- Process restarts by clamping new lagging strand primer
Why does DNA Pol III have to have low processivity?
If it was a highly processive enzyme, it could not release the new Okazaki fragment easily
Does DNA Pol I have low or high processivity?
Low
When can stem-loop structures be formed?
When DNA is denatured and fails to re-anneal properly
What is SSB and what is its importance?
Single stranded DNA binding protein
DNA replication required a supporting cast of SSB
It protects the ssDNA from base pairing and from nuclease
Being displaced by Pol III and replaced as the helix is unwound
Why is most DNA negatively supercoiled?
Easier to replicate
Topoisomerases are used to regulate the degree and type of supercoiling
Arises from unwinding of DNA
How is overwound positively supercoiled DNA turned into underwound negatively supercoiled DNA using Type II topoisomerase?
- Topo II binds to the positive supercoil
- Binding and then cutting BOTH strands of DNA
- Brings coil forward and re-ligates it causing negative supercoil
How is negatively supercoiled DNA relaxed using Type I topoisomerase?
- Topo I binds to negative supercoil
- Cuts one DNA strand
- Causes DNA to unwinds and re-ligates it causing relaxed DNA
How is bacterial DNA polymerisation bi-directional?
2x Pol II complexes enter DNA at an origin of replication
Proceeds in both directions at the same time
Topo IV separates the catenated daughter chromosomes by a double stranded break and relegation
How quickly do eukaryotic DNA Pols polymerise at?
50 nucleotides per second
Why do chromosomes get shorter with each replication?
When DNA polymerase falls off and Okazaki fragments are left so they can join
Primers are erased and gap is filled by DNA polymerase and repaired by DNA ligase on leading strand
Gap on lagging strand cannot be filled by DNA polymerase as there is no primer
How is telomerase a reverse transcriptase?
Telomerase provides an RNA template to synthesise a DNA copy of the template at the 3’ end of the parental lagging strand template
Telomeres are build of repetitive motifs
Where is telomerase active?
Some germline cells
Epithelial cells
Haematopoietic cells
and in >90% of cancer cell lines
- responsible for immortal phenotype of cancer cells
