DNA Replication

Question 1

At what stage of the cell cycle does DNA replication occur?

Answer 1

The S phase

Question 2

What does it mean to say that DNA replication is semi-conservative?

Answer 2

Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strand

Question 3

What does it mean to say that DNA replication is semi-discontinuous?

Answer 3

Due to anti-parallel orientation of double stranded DNA, one strand is 5’ –> 3’ and the other is 3’ –> 5’. Cellular machinery is only able to synthesize new DNA in the 5’ –> 3’ direction, so one strand is sythesized continuously while the other is synthesized in pieces and joined together later.

Question 4

Prokaryotes

How many DNA polymerases?

In what direction do they synthesize? In what direction do they proofread?

Answer 4

7+ different isozymes

5’ to 3’

3’ to 5’

Question 5

What is a processive DNA polymerase? What is a distributive DNA polymerase?

Answer 5

Processive = adds a lot of bases

Distrubutive = adds only a few bases (DNA repair)

Question 6

What is an exonuclease? What is an endonuclease?

Answer 6

Exo: can only degrade from the end of the DNA and work its way in one direction (start at 5’ or start at 3’)

Endo: Can begin to degrade DNA from internal site within the DNA sequence

Question 7

Exonucleases

1. When an exonuclease works in the 5’ to 3’ direction, what is it doing?
2. When an exonuclease works in the 3’ to 5’ direction, what is it doing?

Answer 7

1. Removing primers
2. Proofreading

Question 8

Prokaryotes

• All DNA polymerases require a ___ to initiate replication
• Which DNA polymerases elongate DNA? One of these does most of the elongation, which one is it?
• Which DNA polymerases have 3’ to 5’ exonuclease activity?
• Which DNA polymerase has 5’ to 3’ exonuclease activity?

Answer 8

• Primer
• I, II, III, III
• Proofreading = I, II, III
• Primer removal = DNA Pol I

Question 9

Prokaryotes

• Function DNA Pol I
• Function DNA Pol II
• Function DNA Pol III

Answer 9

• Removes primer, fills gaps after primer is removed, clean up
• Repair
• Principal replication enzyme

Question 10

DNA PolymerasesFunctionProkaryotesEukaryotes Priming DNA A B Synthesis of leading strand C D Synthesis of initial Okazaki fragment strand E F Synthesis of remaining Okazaki G H Gap filling after primer removal I J

Answer 10

A - DnaG primase

B - Pol alpha / primase

C - Pol III

D - Pol delta

E - Pol III

F - Pol alpha/primase

G - Pol III

H - Pol delta

I - Pol I

J - Pol delta

Question 11

What do all DNA polymerases require in order for replication to occur? (2)

Answer 11

1. Primer
2. Template

Question 12

How is a free nucleotide added to the growing chain of DNA?

Answer 12

3’ OH of DNA attacks incoming phosphate of nucleotide, phosphodiester bond formed

Question 13

Prokaryotes

• DnaA protein
• DnaB Protein
• DnaC protein
• HU protein
• FIS protein
• IHF protein
• DnaG protein
• SSB protein
• DNA gyrase

Answer 13

• Recognizes ori sequence (origination), opens duplex at specific site
• Helicase, unwinds DNA
• Helps DnaB bind at origin
• Histone like protein - binds to DNA and stimultes initiation
• Binds to DNA, stimulates initiation
• Binds to DNA, stimulates initiation
• Primase, synthesizes RNA primers
• Single stranded binding protein
• Relieves torsional strain during DNA unwinding

Question 14

Prokaryotes

What is the DUE site?

Answer 14

DNA Unwinding Element (DUE) - area in DNA that is rich in A=T bonds, serves as binding site for DnaA (recognizes DUE site, opens duplex to start replication)

Question 15

Prokaryotes

Describe the process of termination.

Answer 15

2 replication forks meet at sequence called Ter –> trap fork so that it can’t proceed forward any further –> halts DNA Pol III –> final few hundred base pairs are synthesized and replisome dissociates from DNA –> Interlinked circular DNA strands are separted using Topo II

Question 16

What are 4 factors that affect fidelity (the degree of exactness with which something is copied or reproduced) of DNA replication?

Answer 16

1. Thermodynamic factors in base pairing (hydrogen bonds I assume…)
2. Stereospecific effects of DNA polymerization
3. Proofreading
4. DNA Repair

Question 17

Polymerase α Polymerase β Polymerase δ Polymerase ε Polymerase γ Polymerase ζ 3’ to 5’ exonuclease activity essential to nuclear replication Location function

Answer 17

Polymerase α Polymerase β Polymerase δ Polymerase ε Polymerase γ Polymerase ζ 3’ to 5’ exonuclease activity No No Yes Yes Yes No essential to nuclear replication Yes No Yes Yes No No Location Nucleus Nucleus Nucleus Nucleus Mitochondrion Nucleus function Primase Repair Replicase Replicase (lagging strand) Repair

Question 18

Eukaryotes

Function of pol alpha

Answer 18

Lays down the RNA primer and a few DNA nucleotides thereafter, then drops off of template

Question 19

Eukaryotes

Function of Pol delta

Answer 19

Synthesizes DNA strand, has 3’ to 5’ exonuclease activity, REPLICATES EITHER LEADING OR LAGGING STRAND

Question 20

Eukaryotes

Function pol epsilon

Answer 20

Replaces DNA polymerase delta in some situations, such as in DNA repair. DNA polymerase ε may also function at the replication fork, perhaps playing a role analogous to that of the bacterial DNA polymerase I, removing the primers of Okazaki fragments, error correction

Question 21

Eukaryotes

Function Pol Beta and Zeta

Answer 21

Involved in DNA repair

Question 22

1. What is a telomere?
2. What is Telomerase?
3. What results on the DNA after the removal of the last RNA primer on the newly synthesized strand?

Answer 22

1. Long stretches of short repeating sequences (TTAGGG) and preserve integrity and stability of chromosomes by preventing loss of important genetic information each time cells replicate
2. Enzyme that contains RNA molecule that is complementary to repeated sequence found in telomeres. This enzyme binds to the template strand and exhibits reverse transcriptase activty to extend the length of the template strand by thousands of repeats of the telomere sequence. Then, DNA polymerase can come in and bind to the template strand and add complementary bases to the telomere sequence to the newly synthesized DNA strand. Then, when RNA primer is removed from extended DNA, the only information that is being lost is part of the telomere so the coding sequence of the DNA is not distrupted.
3. T-loop - “sticky” end of telomere is looped and tucked under itself to protect telomere ends

Question 23

Prokaryotes use a series of Dna A-G proteins to initiate replication. How is this process different in Eukaryotes?

Answer 23

Eukaryotes have a protein called MCM (minichromosome maintenance) which is a helicase complex that loads onto DNA and performs the functions of Dna A-G, key step in initiation of replication

Question 24

Eukaryotes

Describe the steps in termination of DNA replication

Answer 24

Process is less clear in eukaryotes, mediated by poly-Ub signaling