Class 10 - DNA Replication Notes Flashcards
What are the 3 steps of DNA replication?
Initiation (DNA helicase, topoisomerase, SSBPs, DNA primase)
Elongation (DNA polymerase III, DNA polymerase I, DNA ligase)
Termination (topoisomerase IV (type 2))
What does DNA helicase do? What energy does it use? What is structure?
DNA helicase is a motor protein that uses energy from ATP hydrolysis. It is ring shaped with hexamer subunits
It unwinds the DNA double helix to make a replication fork
How does DNA helicase load on the DNA? Differences in prokaryotes and eukaryotes
In Bacteria DNA melting occurs with ATP (double helix dissociated into single coils) and then ATP is used for helicase to bind and separate strands
In Eukaryotes ATP is used for helicase to load. ATP is used for DNA melting and strands are seperated
How does helicase bind to DNA? How does DNA helicase move in pro and eukaryotes?
DNA helicase binds ATP –> ADP + Pi
In prokaryotes helicase moves 5’ to 3’ on the lagging strand
6 subunits are in the ring
In eukaryotes helicase moves 3’ to 5’ in the leading strand
many more subunits are recruited
How does DNA helicase actually seperate the strands?
It breaks the hydrogen bond between the complementary base pairs
What does topoisomerase do in DNA replication?
DNA topoisomerases are ubiquitous enzymes that are found in all cell types. They act to regulate DNA supercoiling by catalyzing the unwinding of DNA strands. This is done by making an incision in the DNA. There are 2 types of topoisomerases
What is super coiling of DNA? What is it caused by?
if DNA is in a circular form or ends rigidly held then over twisting leads to super coiled states. Super coiling helps relieve helical stress by twisting around itself
What are the 2 types of topoisomerases? How are they different and what energy do they use?
Type 1 creates a single strand which allows the rotation of the other strand (NO ATP)
Type 2 creates a double strand break and then DNA is religated
(requires ATP)
How does the topoisomerase I enzyme work?
Topoisomerase is inactive
When it associated with DNA the enzyme is still inactive because the catalytic tyrosine domain is away from the active site
The catalytic tyrosine domain rotates into the active site and the enzyme becomes active
causes catalysis
What is the mechanism of the cleave if topoisomerase I?
2 Arg groups coordinate the oxygen connected to the phosphate with hydrogen bonds
The water helps the H from the OH-tyrosine leave which creates an attack on phosphorus which then attacks the 5’ Oxygen connected to the deoxyribose
As a result the phosphodiester bond breaks
histidine acts as a positive amino acid and creates hydrogen bond with the 2’ Oxygen and also donates a proton the 5’ O connected to the deoxyribose
What is the mechanism of topoisomerase II in terms of double cleavage?
Tyrosine turns into active site to cleave
Topoisomerase II covalently binds to DNA on both sides and makes a new 5’ end. This preserves the energy of the sugar phosphate DNA backbone. This breaks phosphodiester bonds and creates a 3’OH after the cleavage.
The new 3’OH reacts with topoisomerase II noncovalently
ligation can happen to restore the bond
What is the mechanism of topoisomerase II?
it is believed that an acid-base mechanism is used for DNA cleavage similar to topoisomerase I
D and E amino acids (neg, acidic) coordinate with positive Mg ions. The Mg ions coordinate the negative amino aids and the negative oxygen on the sugar phosphate backbone so they don’t repel.
A base (probably histidine) deprotenates the OH on tyrosine which attacks the phosphate and kicks off the O that connects to a base. This oxygen takes the hydrogen of the acid to create a OH end.
What is the medical application of topoisomerase?
Differences exist between topoisomerases in eukaryotes and prokaryotes
Ciproflaxin is an antibiotic used to treat bacterial infections by interrupting DNA replication and by inhibiting topoisomerase II in prokaryotes
The drug stabilizes cleavage complexes in prokaryotes by inhibiting DNA ligation. This causes many permanent double strand breaks which causes bacterial cell death
Glu/asp and ser stabilize ions in the reaction
What does DNA polymerase require for synthesis of DNA?
needs a 3’ OH group
needs a nucleotide (in triphosphate form)
needs a primer (with 3’ OH)
How does the phosphodiester bond form in DNA replication?
on the new strand
The oxygen of the terminal 3’OH attacks the first phosphate in the incoming nucleotide triphosphate chain. The phosphate attacks the Oxygen in the first phosphodiester bond (connecting to the second phosphate group)
This releases Pyrophosphate (PPi) which seperates into 2 Pi and H20. This drives the reaction where the base is added to the growing strand and 2 phosphates (from the tri chain) leave
What is the mechanism of DNA polymerase in the phosphodiester bond formation?
DNA polymerase has negative, acidic Asp molecules
2 Mg+ ions coordinates Asp with oxygen otherwise they repel
beta and gamma phosphates are distorted around DNA polymerase active site
The catalytic mechanism likely involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues, two of which are highly conserved in all DNA polymerases.
The top Mg2+ ion in the figure facilitates attack of the 3’-hydroxyl group on the FIRST phosphate of the nucleotide triphosphate;
- 2 phosphates leave
the lower Mg2+ ion facilitates displacement of the pyrophosphate.
What are the domains in DNA polymerase?
the fingers, palm, thumb, PHP
PHP is the polymerase and histidinol phosphotase (PHP) domain which is not well understood
DNA fits into those domains
How are Okazaki fragments removed?
The 5’ to 3’ exonuclease activity of DNA polymerase I removes the primer
What does DNA ligase do?
After the RNA primers are removed by exonuclease DNA polymerase I the resulting DNA strand has a nick in the sugar phosphate backbone and this is sealed by DNA ligase4
How is a phosphodiester bond formed?
must be a OH on 3’ carbon
O attacks the first phosphate
and then the bond between the P and the O connecting to the next phosphate group pushes electrons onto that connecting O and 2 phosphates break off
