Replication (I)

Question 1

How are nucleotides added during replication?

Answer 1

Nucleophilic attack of 3’ -OH on alpha-phosphate of incoming dNTP

Question 2

What two factors ensure fidelity during replication?

Answer 2

• Proofreading
• Watson-Crick base pairing

Question 3

Meselson-Stahl Experiment

Answer 3

• Equilibrium density gradient ultracentrifugation
• N-14 and N-15

Question 4

How are the prokaryotic and eukaryotic polymerases distinguished?

Answer 4

Prokaryotic use roman numerals
Eukaryotic use greek letters

Question 5

Who is credited with discovering DNA polymerases?

Answer 5

Arthur Kornberg

Question 6

Where does DNA polymerase add the deoxynucleotides?

Answer 6

The free 3’ -OH on the growing strand.

Question 7

Mnemonic for distuinguishing DnaA, DnaB, DnaC, DnaG

Answer 7

A begins, B breaks, C carries, G gives primers

Question 8

DNA unwinding element (DUE)

Answer 8

An AT rich sequence near the OriC that is prone to unwinding.

Question 9

In which domain is the DNA unwinding element (DUE) seen?

Answer 9

Prokaryotes

Question 10

What is encompassed by “prepriming” in prokaryotes?

Answer 10

DnaA binding and DNA wrapping, DUE unwinding
DnaB hexamers binding to strands of open DNA

Question 11

What protein name is associated with DnaB?

Answer 11

Helicase

Question 12

What does DnaC do?

Answer 12

It acts as a “clamp loader” - pries open the DnaB, allowing for DNAinsertion and helicase opening

Question 13

In what domain is the primosome seen?

Answer 13

Prokaryotes (Primosome = Prokaryote)

Question 14

What are the components of the primsome?

Answer 14

• DnaG (primase)
• DnaB
• 5 other proteins

Question 15

What polymerase does most of the synthesis for prokaryotes?

Answer 15

DNA pol III

Question 16

What is a consideration regarding primer formation on the lagging strand?

Answer 16

• Primosome has to initiate each Okazaki fragment
• Primosome gets moved in the 5’ –> 3’ direction on the template strand, so it must be briefly reversed to lay down the primer

Question 17

In what domain is the replisome found?

Answer 17

Prokaryotes (PRokaryotes = Primosome, Replisome)

Question 18

What are two proteins that are not part of the replisome, but still needed for elongation?

Answer 18

• DNA gyrase
• SSBs

Question 19

In which domain are SSBPs found?

Answer 19

Prokaryotes

Question 20

Holoenzyme

Answer 20

An enzyme with its required cofactor(s).

Question 21

Contents of the Pol III holoenzyme

Answer 21

• 2 catalytic cores
• Linked gamma complex (“clamp loader”)
• Beta clamp on each core

Question 22

What is the function of the beta clamp?

Answer 22

It helps the catalytic cores stay attached to the DNA

Question 23

What is the process of loading the beta clamp?

Answer 23

• Clamp loading complex binds to 3 ATPs, Beta-clamp dimer
• ATP binding forces the beta clamp to open
• DNA enters the complex
• ATP hydrolysis causes the clamp to close around the DNA

Question 24

What is the name of the process performed by DNA Pol I?

Answer 24

“Nick translation”

Question 25

What does DNA Pol I do?

Answer 25

Removes RNA primers by excising RNs and replacing them with dRNs - The nick gets moved to the 3' end of the DNA that has replaced the Okazaki fragment

Question 26

What is notable about the structure of DNA ligase?

Answer 26

- Hydrophobic pocket for NAD or ATP - Cleft for DNA binding

Question 27

What is the basic process used by DNA ligase?

Answer 27

- Adenyl group is moved from lysine to 5' phosphate of the nick - 3' -OH attacks the 5' phosphate to close the gapW

Question 28

What does the Tus-Ter complex do?

Answer 28

Terminates replication by acting as a "roadblock" that traps the replication fork

Question 29

Ter Sequence

Answer 29

A 20 bp segment opposite the OriC

Question 30

Tus

Answer 30

Terminus Utilization Substance

Question 31

What domain is associated with the Tus-Ter complex?

Answer 31

Prokaryotes

Question 32

What does Topo IV do?

Answer 32

Separates catenanes at the end of prokaryotic replication

Question 33

What class of Topo does Topo IV belong to?

Answer 33

Top II

Question 34

What are three major ways that eukaryotic replication differs from prokaryotic replication?

Answer 34

- Requires multiple DNA polymerases - Multiple origins of replication - Telomeres

Question 35

In what stage of the cell cycle is DNA synthesis initiated?

Answer 35

Late G1

Question 36

What controls cell cycle checkpoints?

Answer 36

CDKs and cyclins

Question 37

With what domain is the pre-replication complex associated?

Answer 37

Eukaryotes

Question 38

What is the eukaryotic equivalent of DnaA?

Answer 38

ORC (Origin recognition complex), 6 proteins bound to DNA

Question 39

What causes the conformational change in the ORC?

Answer 39

ATP hydrolysis

Question 40

What happens when the ORC undergoes the conformational change

Answer 40

cdc6 can bind (it must be phosphorylated by CDK)

Question 41

What is the eukaryotic equivalent to DnaB?

Answer 41

MCM proteins

Question 42

What is the other name for MCM proteins?

Answer 42

Licensing proteins

Question 43

How do helicases move?

Answer 43

ATP hydrolysis

Question 44

What are the two proteins needed to activate the pre-replication complex?

Answer 44

- cdc45 - GINS