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
What are the roles of the initiator?
– initiator binds DNA
– Initiator unwinds ( a little) DNA at the origin
– Initiator recruits other replication proteins: helicase, primase and the Pol III complex
– binds to AT rich site because there’s only 2 hydrogen bonds here where as at CG there are 3 hydrogen bonds
– Eukaryotes initiate replication at sites that have an AT-rich sequence
What are end replication problems in Eukaryotes?
– the need to use a primer to initiate DNA synthesis followed by removal of primer prevents complete replication of one strand at each end of DNA
– this will cause progressive shortening of DNA after each round of DNA replication
– Eukaryotic cells use a special enzyme called telomerase to replicate the ends completely
Why can’t Pol I fill in the gaps during DNA synthesis?
– there is no 3’ hydroxyl group so Pol I doesn’t have anything to attach to
– can’t do it’s nucleophilic attack
Describe the function of telomeres.
– telomeres act as chromosome caps. They bind proteins that protect the ends of chromosomes from exonucelolytic digestion or end-joining
– in most eukaryotes, the ends of linear DNA molecules are replicated by a unique mechanism using enzyme telomerase, that synthesizes short DNA repeats
– protecting open region from enzymes –> not an actually physical cap but a sequence that protects
Describe the synthesis of Telomeric DNA.
– Telomerase carries and uses an DNA template for DNA synthesis (reverse transcriptase)
– carries it’s own RNA template
– complementary to end of telomeric DNA (hybridizes)
– the 3’ end of DNA is elongated, using RNA as a primer
– telomere is elongated multiple times
– other strand is synthesized by normal lagging strand synthesis
What extends the top strand of DNA?
– DNA pol 3 will come in and extend
– we need primer to come on and Pol 3 starts adding bases
Describe how primase and DNA Polymerase extend the 5’ End.
– after telomerase has extended the 3’ end, primase and DNA polymerase can carry out replication using the new, longer end as a template
– DNA Pol III adds the new bases and DNA Pol I removes the RNA primer
How does Telomere stabilize chromosome ends?
– also regulates telomere lenght
– telomere binding proteins protect the end from degradation by exonucleases, recombination, ligation, etc.
– as telomere is lengthened, more telomere binding proteins bind and inhibit telomerase
– as telomere shortens, reduced binding of telomere binding proteins allows telomerase to bind and lengthen telomere
Describe Telomerase Activity in Animals.
– Telomerase is inactive in most somatic cells.
– Telomerase remains active in germ line and in stem cells.
– Telomerase is reactivated in immortalized (tumor) cells
– Mice lacking telomerase are less likely to form certain types of tumors
– associated with the idea of regulating aging
Describe how DNA replication is used for sequencing reactions.
– normally you’ll have a DNTP come in but occasionally you’ll have one of these ddATP, ddCTP, ddGTP, ddTTP come in because there’s so many replication cycles happening which terminates the reaction
– all of the dds have 3’ hydrogen group which is a terminating sequencing base —> meaning the DNA replication will just stop
T or F, in normal replication reaction we have DNTP
True; in sequencing reaction we’re going to use DNTP
– DNTP have 3’ hydroxyl group in order for polymerase to come in and bind and add DNA bases
Describe DNA Sequencing Gel.
– Four different reactions are performed, each with a different dideoxynucleotide, ddA, ddC, ddG and ddT
– each reaction is run on a denaturing polyacrylamide gel, which allows analysis of the single synthesized strand with single base resolution
– primer was radioactive or fluorescent and can be visualized on the gel
– the position of a band in a lane indicates where each base was incorporated
Describe High-Throughput DNA Sequencing.
– more recently, standard dideoxy sequencing reactions are carried out in large batches (96 or 384 at a time) on machines that can separate samples on re-usable columns (96 or 384) instead of gels
– the sequencing reactions are carried out all together in a single reaction that has all four of the normal dNTPs and a low concentration of each of the dideoxy-NTPs, each being labeled with a different fluorescent-colored dye
– the colored DNA molecules are detected as they pass the end of the column, reading out the sequence
