Lecture 31 (add sequence portion) Flashcards
What are the three types of DNA coiling?
Negative, positive, and relaxed
What type of coiling do prokaryotes and eukaryotes normally have?
Negative supercoiling
What does negative supercoiling do to DNA? How does it prepare it?
Negative supercoil prepares the DNA for the replication process which requires separation of the DNA strands
What is coiling in the opposite direction?
negative supercoiling
What is DNA coiled in the same direction?
Positive coiling
What type of coiling do bacterial genomes have?
Negative supercoiling
At what bp does replication occur in E. Coli?
100,000 bp/min
What is the enzyme that is responsible for unwinding the double helix strand of DNA?
Helicase but there are other enzymes ahead of helicase to remove positive supercoils. Helicase also generates supercoils ahead of the replication fork
What other enzymes relieve positive supercoils that are NOT helicase?
Topoisomerase I and II
What does topoisomerase do?
Unwind the double helix during DNA replication. These work ahead of the fork
What does topoisomerase I do?
can make single-stranded breaks to relax the helix.
What does topoisomerase II do?
It contains gyrase and is able to break and rejoin the double stranded DNA
How does topoisomerase Iwork? How does it bind?
It binds to one strand of DNA, cuts it, and the DNA can then swivel and relieve tension.The intact strand of DNA passes through the nick resulting in the relaxation of the torsional strain. Then, it forms a phosphodiester bind.
What is true about topoisomerase I and energy?
It does not need ATP but instead transfers the phosphodiester bind from the DNA to the enzyme and back again. Does NOT hydrolyze
What is true about topoisomerase and energy use?
It requires ATP
How does topoisomerase II work? How does it bind?
It uses ATP to cut both strands, passes the molecules through the cut section, and reseals the phosphodiester bond. It cleaves the strand and generates a negative coil. Relieves pos coil and introduces negative coiling
What happens when coiling is too tight?
DNA can’t unwind for replication
How do eukaryotes differ from prokaryotes during DNA replication?
Since they are linear, they do not rotate freely, also includes their binding to proteins.
What relieves coiling in eukaryotes?
Topoisomerase I or II
Why isn’t gyrase needed in eukaryotes?
Because gyrase introduced negative coils which is something that eukaryotes already have, they just need positive coils to be relieved
What happens when topoisomerase is inhibited?
They are useful because they stop DNA replication, stop reading DNA for protein produc, and stop repair of DNA damage
Why is topoisomerase inhibitors useful for cancer treatment?
It is useful because they stop replication in target cells that are replicative
How do topoisomerase inhibitors work and benefit antibacterial drugs?
Inhibitors are helpful because they target prokaryotic topoisomerase and stop bacterial DNA replication and leaves eukaryotes topoisomerase untouched
What are the antibacterials targeted by inhibitors?
nalixidic and ciprofloxacin
What are the cancer treatments targeted by the inhibitors?
camptothecin, doxorubicin, and mitoxanthrone
How many Pol does E. Coli have?
3 one in the leading strand, and two for the lagging strand
What form does DNA need to be during DNA replication?
Negative coiling and chromatin must be dismantled prior to replication and reform daughter strands
What controls replication?
Replication is based on DNA sequence on the Ori site and then binds to the initiator protein
What does the initiator protein do?
regulate the replication of DNA
Where does DNA synthesis begin?
At the origin which is adjacent to the replicator
What are the roles of the initiator?
1.Initiator binds DNA
2. Initiator unwinds (a little)
DNA at the origin
3. Initiator recruits other
replication proteins:
helicase, primase and the
Pol III complex
Where do eukaryotes initiate replication?
sites that have an AT-rich sequence and helicase unwinds because of hydrogen bonds
What are some problems that arise in eukaryotes?
The needing to use a primer for DNA synthesis and then removing them causes replication to be complete in one stand. This causes for shortening in the strands over time after each round of replication.
What enzyme do eukaryotes use to replicate ends completely?
telomerase
What cells can extend their ends?
Germ(sperm and egg) and stem cells
What are telomeres?
Repeat and non-coding
How do telomeres work?
They work as chromosome caps and bind to proteins that protect the ends of chromosomes from end-joining and exonucleolytic digestion
What is telomerase? How does it work?
It is a unique mechanism that synthesizes short DNA repeats
What template does telomerase use?
RNA template for DNA synthesis(reverse transcriptase). Dependent on DNA polymerase
What is true about telomerase and primers?
It has its own primer that allows extending. This first starts in the 3’ end of DNA using the RNA primer
What happens when telomerase has extended the 3’?
Primase and DNA
polymerase can carry out
replication using the new,
longer end as a template
What does DNA Pol III do?
adds the new bases
What does DNA Pol I do?
DNA Pol I removes the
RNA primer
What does telomere binding proteins do? What type of protection does it have?
It protects from degradation by exonucleases, recombination, ligation
What happens when telemore is lengthened?
more telomere binding
proteins bind and inhibit
telomerase
What happens when telomere shortens?
reduced binding of telomere binding proteins allows telomerase to bind and lengthen telomere
When is telomerase inactive?
somatic cells
When is telomerase activated?
Immortalized (tumor) cells in cancer and activates multiple replication cycles
