Lecture 9 Flashcards
How is helix first separated at Ori sequence?
Initiation factor DNAa recognizes and binds 9-mer repeats, then separates the DNA at 3AT-rich tandem repeats
Which protein unwinds the helix as replication occur?
DNAb helicase complexes proceed bidirectionally
How is re-annealing prevented in prokaryotes and eukaryotes?
Single Strand DNA-binding protein (SSB) in prokaryote
Replication Factor A in eukaryote
What does DNA polymerase need for for replication (5)?
- all four dNTPs
- fragment of DNA that acts as template
- Magnesium ions
- primer providing a free 3’-OH group
- A sliding clamp
Why is RNA less stable than DNA?
RNA has 2’-OH
Direction of DNA synthesis and template reading
5’-3’ for synthesis
3’-5’ for reading
Why does dNTP need to be in the form of triphosphate?
it gives energy for formation of phosphodiester bond.
Why is sliding clamp important during DNA replication?
DNA pol assembles with clamp to form a highly processive enzyme. Without it, DNA pol would dissociate after each nucleotide synthesis, and have to have primer again to reattach to the template.
Which enzyme remove primer in Okazaki fragments?
Ribonuclease
Why does ligase have to join okazaki fragments not DNA pol?
Because DNA polymerase can work only with triphosphates, but fragment is monophosphate.
What is the difference between DNA polaymerase I and III? (2 same function, 1 different function)
Both of them synthesize nucleotide,
Both of them have 3’-5’ exonuclease activity (direct repair). When there is mistake, pol pauses, and 3’end of strand is transferred to exonuclease site, for removal of mispaired base.
but Type I has 5’-3’ exonuclease activiy which function as ribonuclease. So it can remove primers and replace it with nucleotides at the same time. so it removes RNA primers of Okazaki fragment and elongates the previous Okazaki fragment.
What is role of DNA gyrase?
relieve positive supercoils ahead of the fork.
Both strand of dsDNA is cut, and another duplex DNA is then passed through the cut ends and the ds break resealed.
What is the name of protein used for replication termination?
Tus protein
What is the role of Tus protein
it binds tot he Ter sequence, then prevents DNA duplex from unwinding by blocking the replication fork and inhibiting DNAb (helicase), so it is called contrahelicase.
After termination, resulting progeny chromosomes are interwined, so what is done to resolve this?
Gyrase makes cut in dsDNA to unwind them.
Why azidothymidine (AZT) used in treatment of HIV?
AZT is analogue of thymidine, so it gets incorporated into reverse transcription process. Since it does not have 3’OH (it has 3’N), it terminates reverse transcription process. (Since it doesn’t have 3’OH synthesis of next nt is not possible by DNA pol)
Why is didanosine used for treatment of viral infection?
DDI has hydrogen replcing 3’-OH on sugar. Thus terminating the reverse transcription process.
What is the advantage of using didanosine?
it has much higher affinity to viral reverse transcriptase than human polymerase. So drug is much more specific.
What is side effect of didanosine?
it has effect on gamma polymerase in mitochondria so side effect would lack of ATP
Why is tenofovir used for viral infection?
it is deoxyadenosine 5’triphosphate analogue. Tenofovir is phosphrylated by host kinase and gets incorporated into reverse transcription process. It lacks 3’-OH on sugar.
Why is acyclovir used for viral infection?
It also lacks 3’-OH, and is analogue of deoxyguanosine. , causing termination of viral DNA synthesis.
Why is acyclovir advantageous?
It needs to be phosphorylated by kinase before incorporation into viral DNA, but viral thymidine kinase is much more effecive in catalysing 1st phosphorylation than host thymidine kinase, so acyclovir is only activated in infected cells, thus being more potent against viral DNA polymerase than host.
What diseases is acyclovir highly specific? (2)
Herpes simplex and varicella zoster
How does Gyrase inhibitor behave?
it prevents re-ligation of the cleaved DNA duplex by DNA Gyrase, which turn into permanent DNA strand breaks causing cell death of bacteria.
Name gyrase inhibitor based antibiotics
ciprofloxacin
What is the drug family of hyrase inhibitor
Fluoroquinoloes, quinolones
What is the difference between topoisomerase I and II
I : make a cut in only one strand of ds DNA
II: make a cut in both strands of DNA
Which enzyme has reverse-transcriptase activity during DNA replication in eukaryote?
telomerase
What is the role of DNA polymerase alpha?
priming during replication initiation
What is the role of DNA polymerase gamma? (2)
lagging strand synthesis (including gap filling)
also has 3’-5’ exonuclease
what is the role of DNA polymerase ebsylon (2)
leading strand synthesis
has 3’-5’ exonuclease
Which enzyme is responsible for removal of RNA primer during DNA replication in eukaryotes? (2)
RNase H or Flap endonuclease 1 (FEN1)
What is the effect of cytosine arabinoside (araC)
Termination of elongation
For what disease is araC used?
Leukemia, lymphoma
What is the mechanism of araC
It has 3’OH that allows further chain elongation when incorporated, however it has another OH at position 2, which prevents polymerase adding the next dNTP due to stereo restraints, which leads to termination.
What is the mechanism of araA
It has 3’OH, but planar configuration of the arabinoside prevents DNA elongation due to OH at position2.
What is araA used for?
for treatment of relapsed childhood acute lymphoblastic leukaemia (anti-neoplastic agent)
what are 2 drugs that interfere with eukaryotic topoisomerases?
etoposide, camptothecin
What is the name of group of drugs that interfere eukaryotic topoisomerases?
topotecan
For which disease is topotecan used?
ovarian cancer, lung cancer, acute myelocytic leukemia
Difference between etoposide and camptothcin
etoposide target topoisomerase II, camptothecin target topoisomerase I
What is the mechanism of topotecan?
traps the enzyme topoisomerase when it cleaves DNA causing double strand breaks, preventing DNA replication
What is actinomycin D?
Inhibitors of replication
What is the mechanism of actinomycin D and its effect (2)
It binds to G-C rich region of DNA. Then, prevents formation of regions of ssDNA.
Also, inhibit RNA transcription (cell needs synthesis of specific RNA’s and protein for cell cycle)
What disease is actinomycinD used for?
Wilm tumor, Kaposi sarcoma, testicular cancer
