LECTURE 31 - FINAL Flashcards
Describe supercoiling during replication.
– DNA can be positively supercoiled, relaxed, or negatively supercoiled
– prokaryotes and eukaryotes usually have negatively supercoiled DNA
– Negative supercoil prepares the DNA for the replication process which requires separation of the DNA strands
What happens to the DNA during bacterial replication?
– During bacterial replication the double-helix must be unwound so that each strand may serve as a template for replication to a daughter strand
– chromosome is circular –> no free ends to relieve tension when parental strands unwind
– DNA duplex ahead of replication fork becomes over-wound and region behind replication fork becomes under wound
– both of these put DNA under tension
– as more and more DNA becomes unwound, tension of regions would inhibit replication fork from moving forward, thus mechanism is in place to release tension and allow replication fork to proceed
How is DNA supercoiling dealt with during DNA unwinding?
– Helicase –> primary enzyme involved in unwinding double-stranded DNA
– also have enzymes present ahead of helicase to remove positive supercoils so replication can progress
– enzymes known as Topoisomerases
– Top I –> make single-stranded breaks to relax helix
– Top II (gyrase) –> break and rejoin double stranded DNA
Describe the mechanism of Top I.
– Type I cut only one strand of DNA, whereas Type II cuts both strands
– when Type I binds to one strand of DNA and cuts it, DNA can then swivel on other strand relieving tension. Intact strand of DNA passes through nick resulting in relaxation of torsional strain
– Topo I can then reform phosphodiester bond, creating unnicked, double-stranded DNA molecule again and then disassociates from DNA molecule
– Top I enzymes don’t utilize any energy in this process, they simply transfer phosphodiester bond from DNA to enzyme and back again
Describe the mechanism of Top II.
– called gyrase and most common topoisomerase in E. coli
– Circular chromosome keep DNA from rotating freely.
– Positive supercoils rotate to the right, negative to the left.
– When coils get too tight, the DNA can’t unwind for replication.
– Gyrase cuts both strands, relieves positive supercoil and introduces a negative supercoil.
– Requires ATP energy.
Why don’t eukaryotes need gyrase?
Eukaryotes don’t need gyrase because our chromosomes are linear and our DNA has a natural negative supercoil with the histone.
prefer negative coil during replication because negative coiling is opposite of right-handed —> twisted to left
if there’s a positive helix, you would twist the DNA into more of a right-handed fashion —> which would knot up the DNA
negative coils are easier to unwind
Why is a negative supercoil preferred during replication?
prefer negative coil during replication because negative coiling is opposite of right-handed —> twisted to left
if there’s a positive helix, you would twist the DNA into more of a right-handed fashion —> which would knot up the DNA
negative coils are easier to unwind
How do Eukaryotes deal with supercoiling?
– even though chromosomes are linear –> do not rotate freely during DNA replication because of many initiation sites of DNA replication and their great length (same issues with supercoiling)
– when DNA is complexed w/ histone proteins this introduces areas of local negative supercoiling –> compensated by positive supercoils in other areas
– positive supercoils relieved by either Topo I or Topo II enzymes and net effect is DNA is negatively supercoiled bc of areas wrapped around histones
– bc of this eukaryotes don’t need enzyme to introduce negative supercoils like gyrase
– simply need something to relieve positive supercoils, allowing negative supercoils to function to counteract tension that will be introduced by DNA helicase as it’s unwound in replication fork
What can happen when we don’t have topoisomerase or when it’s defective and can’t work?
– can’t have DNA replication
– DNA can’t unwind so no replication can happen bc there are no free bases
– this means that topoisomerase do a lot and play a large role
Describe drugs that inhibit Topoisomerases.
– molecules that inhibit toposiomerases can be useful as drugs since halting action of topoisomerase stop DNA replciation, stop reading of DNA for protein production and stop repair of DNA damage
– Topoisomerases have been used for cancer treatment because of their ability to stop replication in target cells that are highly replicative –> like cancer cells
– can be useful antibacterial drugs –> can be designed to selectively target prokaryotic topoisomerase, thus stopping bacterial DNA replication, while leaving eukaryotic topoisomerases untouched
What protein machinery is present at the replication fork of prokaryotes?
– Helicase unwinds DNA (ATP)
– Primase is attached to it, makes primers, called
Primasome.
– Single-stranded binding protein keep DNA strands unwound.
– In E. Coli, there are 3 Pol III molecules, one for leading strand and two for lagging strand.
– The two sides have the same core enzyme, but lagging strand has extra subunits (not shown). Together called the holoenzyme.
Describe the holoenzyme.
– the hollow enzyme consists of many diff enzymes.
– not apart of it is DNA ligase and DNA polymerase
– 2 polymerase 3 on the lagging strand.
What is the function of Pol I?
– removes primer and adds DNA bases
T or F, chromatin must be dismantled prior to replication and reform on daughter strands
True
Describe Initiation of DNA replication: E. Coli.
– replication is controlled by a DNA sequence called the origin of replication that binds an “initiator” protein
– the initiator protein regulates replication of the DNA
– DNA synthesis actually begins at the origin, which is usually within or adjacent to replicator
