Lecture 9-DNA Synthesis (Nakai) Flashcards
What are 3 diseases involved in repeated trinucleotides?
- Fragile X syndrome
- Huntingtons
- Myotonic Dystrophy
How do sites of origin differ between prokaryotes and eukaryotes during chromosomal replication?
prokaryotes: 1
eukaryotes: many, some are activated at different times within S phase
How do the speed of replication differ between prokaryotes and eukaryotes?
Prokaryotes replication fork seems to move much faster than eukaryotes, probably due to the higher fidelity of replication in eukaryotes.
which protein synthesizes the primer on leading/lagging strands? which one synthesizes the DNA?
- primase
- DNA pol
How do prokaryotic okazaki fragments compare to eukaryotic?
they are much smaller!
In which direction are DNA and RNA synthesized?
- 5’ –> 3’
During DNA polymerization, with incorrect bping there is a ______ Km and _____ Vmax. How does this differ from correct bping?
Incorrect:
- high Km
- low Vmax
Correct:
- low Km
- high Vmax
Nucleotide discrimination
the nucleases at the replication fork are highly discriminator
- one method of ensuring high accuracy of replication
Which DNA pol proofreads?
DNA pol I
What are the functions of DNA Pol I?
- proofreading
- exonuclease: removes RNA primer from okazaki fragments
- fills in the gap from RNA primers of okazaki fragments with LOW PROCESSIVITY which makes it good for this purpose
Generally explain the function of exonucleases, specifically DNA Pol I.
- exonucleases work from the end of DNA hydrolyzing the phosphodiester bonds of the backbone and thus cannot work on circular DNA
- THEY WORK 3’-5’!!
How would DNA Pol I recognize a mismatched C-A bond and why would this match happen in the first place?
- C undergoes tautomeric shift into a high energy state and bonds with A
- C will eventually convert back to its lower energy form and will stay in a “frayed” position
- This frayed form enters the exonuclease site of DNA Pol I
- mispaired 3’OH of C is recognized by the Pol and it slides back so the mispaired base is in the 3’-5’ exonuclease site of the Pol
- nucleotide removed
- Pol continues forward (it moves in 5’-3’ position, the direction of synthesis but will work in the 3’-5’ direction)
Describe the dimeric DNA Pol III
- Has 2 DNA Pol III’s associated with it
- 1st: leading strand synthesis: highly processive
- 2nd: lagging strand synthesis, forms loop structure and accustomed to many attachments and detachments
- -> doesn’t have to leave the complex to scan for the next primer
Describe the DNA Pol complex at the replication fork (5)
- dimeric DNA Pol
- helicase associated with it
- SSBs that keep the single stranded DNA from collapsing on itself
- exonuclease activity that proofreads the DNA 3-5’
- primase
What cuts the DNA that becomes supercoiled to relieve tension?
gyrase (topoisomerase)
Which polymerases are associated with primase and lagging strand synthesis?
- only pol alpha!
Which pols are associated with propogation of the replication fork
- alpha
- delta
Which pols are thought to be synonymous with Pol III?
- delta
- epsilon
What are the features of the origin of replication?
- 3 tandem 13bp repeated consensus sequences (AT rich, upstream of:
- 4, 9bp consensus sequences for binding of DnaA protein which regulates when replication will be initiated
DnaB
prokaryotic helicase associated with replication fork machinery (DNA Pol III dimer, SSB, primase)
DNA ligase requires _____ to function
ATP or NAD+
Roughly delineate the process of unwinding DNA at the origin
- DnaA binds the wound DNA (ATP requiring) and begins unwinding of the duplex
- DnaB helicase binds to the unwinding duplex and, with the help of ATP and DnaC will begin unwinding the strands further
The major event in initiation is ______(3).
- Duplex opening,
- loading the strands
- laying down the primer
Explain how eukaryotes license
- ORC stays bound most of the time (=DnaA)
- Cdc6/18 and Cdt1 (=DnaC) are the molecular matchmakers that recruit MCM (=DnaB)
- this complex is considered licensed.
How does licensing change throughout the cell cycle.
- G1: ends with the licensing complex forming (meanwhile proteins and RNA are being synthesized)
- S: licensed complex allows for synthesis of DNA. Ends with Cdc6/18 leaving and being degraded, Cdt1 is sequestered by geminin
- G2/M: geminin is present which both prevents Cdc6 to be synthesized and also keeps Cdt1 sequestered. At the end of M geminin is degraded so licensing can recur.
G0 cells do not have the ability to _______ and therefore ______.
- license
- enter a stage of senesensce
