Lecture 9-DNA Synthesis (Nakai)

Question 1

What are 3 diseases involved in repeated trinucleotides?

Answer 1

• Fragile X syndrome
• Huntingtons
• Myotonic Dystrophy

Question 2

How do sites of origin differ between prokaryotes and eukaryotes during chromosomal replication?

Answer 2

prokaryotes: 1
eukaryotes: many, some are activated at different times within S phase

Question 3

How do the speed of replication differ between prokaryotes and eukaryotes?

Answer 3

Prokaryotes replication fork seems to move much faster than eukaryotes, probably due to the higher fidelity of replication in eukaryotes.

Question 4

which protein synthesizes the primer on leading/lagging strands? which one synthesizes the DNA?

Answer 4

• primase

- DNA pol

Question 5

How do prokaryotic okazaki fragments compare to eukaryotic?

Answer 5

they are much smaller!

Question 6

In which direction are DNA and RNA synthesized?

Answer 6

• 5’ –> 3’

Question 7

During DNA polymerization, with incorrect bping there is a ______ Km and _____ Vmax. How does this differ from correct bping?

Answer 7

Incorrect:

• high Km
• low Vmax

Correct:

• low Km
• high Vmax

Question 8

Nucleotide discrimination

Answer 8

the nucleases at the replication fork are highly discriminator
- one method of ensuring high accuracy of replication

Question 9

Which DNA pol proofreads?

Answer 9

DNA pol I

Question 10

What are the functions of DNA Pol I?

Answer 10

• 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

Question 11

Generally explain the function of exonucleases, specifically DNA Pol I.

Answer 11

• 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’!!

Question 12

How would DNA Pol I recognize a mismatched C-A bond and why would this match happen in the first place?

Answer 12

• 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)

Question 13

Describe the dimeric DNA Pol III

Answer 13

• 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

Question 14

Describe the DNA Pol complex at the replication fork (5)

Answer 14

• 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

Question 15

What cuts the DNA that becomes supercoiled to relieve tension?

Answer 15

gyrase (topoisomerase)

Question 16

Which polymerases are associated with primase and lagging strand synthesis?

Answer 16

• only pol alpha!

Question 17

Which pols are associated with propogation of the replication fork

Answer 17

• alpha

- delta

Question 18

Which pols are thought to be synonymous with Pol III?

Answer 18

• delta

- epsilon

Question 19

What are the features of the origin of replication?

Answer 19

• 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

Question 20

DnaB

Answer 20

prokaryotic helicase associated with replication fork machinery (DNA Pol III dimer, SSB, primase)

Question 21

DNA ligase requires _____ to function

Answer 21

ATP or NAD+

Question 22

Roughly delineate the process of unwinding DNA at the origin

Answer 22

• 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

Question 23

The major event in initiation is ______(3).

Answer 23

• Duplex opening,
• loading the strands
• laying down the primer

Question 24

Explain how eukaryotes license

Answer 24

• 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.

Question 25

How does licensing change throughout the cell cycle.

Answer 25

• 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.

Question 26

G0 cells do not have the ability to _______ and therefore ______.

Answer 26

• license

- enter a stage of senesensce