Lecture 9 Flashcards
What are the required components for DNA synthesis
- all four dNTPs (A,T,C,G)
- a fragment of DNA to act as a template
- DNA polymerase
- Mg ions
- a primer providing a free 3’ -OH group
What are the drugs Zidovudine and Didanosine?
They are both anti-HIV drugs that will be Incorporated into the DNA chain
However termination occurs because both of these drugs are nucleosides and do not have a 3 prime OH group
Explain acyclovir?
Is a DNA polymerase inhibitor
Lack of a 3 prime OH group
used to treat herpes simplex
Explain tenofovir?
A drug used to treat HIV
This drug competes with adenosine
This compound has no sugar, thus no OH group
this leads to termination of DNA synthesis
What is cytosine arabinoside?
This compound does have a 3 prime OH group
BUT the OH group is not positioned correctly
This leads to chain termination
adenosine arabinosid works in a similar way
How can the cell fix a base misincorporation?
DNA poly has a 3 to 5 prime proofreading exonuclease
will fix on the 3’ OH end
What are the players of DNA replication in prokaryotes ?
- DNaA protein (initiator protein)
- DNA helicase
- Helicase inhibitor
- DNA primase
- DNA poly I
- DNA poly III
- single stranded binding protein
What is the role of DNaA protein during prokaryotic DNA replication?
Binds to the origin of replication and breaks hydrogen bonds between the bases
What is the role of DNA helicase protein during prokaryotic DNA replication?
opens and binds primase to form primosome?
What is the role of the helicase inhibitor (prokaryote)?
delivers helicase to the DNA template
Role of DNA primase (prokaryote)?
an RNA polymerase that will synthesize a RNA primer on the lagging strand to enable DNA poly to synthesize DNA
role of DNA poly I (prokaryote)?
remove RNA primer and replace with DNA
role of DNA poly III (prokaryote)?
synthesis of leading and lagging strands
Role of ssb?
binds to single stranded DNA in the replication bubble and prevents it from re-annealing
Model of initiation of replication at E.coli OriC?
- DNaA proteins will bind to the 9-mers at the origin
- strand separation occurs at the region of the 13-mers
- Helicase inhibitor protein delivers helicase
- helicase clamps around each strand of DNA
- Helicase will unwind the DNA
How to prevent single stranded DNA from reanneling (prokaryotes)?
SSB proteins
How are the okazaki fragments joined together in prokaryotes?
DNA ligase
How is the DNA poly III loaded and maintained on the single stranded DNA template?
Clamp proteins.
these proteins hold DNA poly onto the template for synthesis
How can positive supercoiling be reversed?
topoisomerase enzymes
What is the role of DNA gyrase (prokaryotes)?
This enzyme introduces negative supercoils into the DNA…. this reduces positive supercoiling by opening the DNA
What is ciprofloxacin?
this is a drug that inhibits bacterial DNA gyrase
used to treat respiratory and urinary tract infections
What are the eukaryotic DNA polymerases?
Polymerase alpha
polyermase theta
What is the role of polymerase alpha (eukaryotes)?
synthesizes RNA primer on the leading and lagging strand
subunit possesses primase activity and initiates DNA synthesis
low processivity- falls off after short segment
no exonuclease activity
What is the role of polymerase theta (eukaryotes)?
Synthesis DNA from leading and lagging strands
main replicative enzyme
high processivity- can synthesize long stretches of DNA
3 to 5 exonuclease activity
Explain camptothecin?
this is an anti cancer drug that binds to and inhibits the activity of topoisomerase I in eukaryotes
this leads to DNA breakage
Explain eptoside?
anti cancer drug that inhibits the activity of topoisomerase II
What does actinomycin D (dactinomycin) do?
prevents DNA replication
prevents the formation of regions of ss DNA
inhibits RNA transcription
Differences between prokaryote and eukaryote DNA replication
Prokaryote- only one origin of replication
DNA poly I and III
primers are removed different (DNA poly I)
eukaryote- multiple origin of replication
Polymerase alpha and theta