Chapter 20: DNA Replication

Question 1

What is the building block of DNA and RNA?

Answer 1

Nucleotides consisting of nitrogenous base, ribose/deoxyribose sugar, and Phosphoryl group

Question 2

Which bases are purines and which are pyrimidines? What are the key differences?

Answer 2

Purines: A and G
- Double rings

Pyrimidines C,T,U
- Single rings

• AT form 2 H bonds, CG forms 3 H bonds

Question 3

What is DNA replication?

Answer 3

• The process of creating copies of DNA
• Semi-conservative
• Begins at origin, proceeds bidirectionally
• Synthesized in a 5’ ->3’ direction (semi-discontinuous)

Question 4

How was DNA tested to be semiconservative?

Answer 4

Meselson-Stahl
- DNA was grown in a N15 medium, transfered to a N14 and the new DNA had a N14 and N15 strand

Question 5

Compare a template and a primer.

Answer 5

Template:
- Single unpaired DNA strand

Primer:
- Pre-existing short of DNA or RNA
- Provides free hydroxyl group at 3’ end which is where dNTPs are added

Question 6

Describe the polymerase mechanism of DNA polymerase.

Answer 6

• Catalysis involves 2 Mg2+ ions at active site
• One Mg2+ ion helps deprotenate 3’ OH group making it a better nucleophile
• The 3’ OH attacks the Phosphate from incoming dNTP
• Other Mg2+ binds incoming dNTP and stabilizes negative charge

Question 7

What is Processivity?

Answer 7

• The average number of nucleotides added before a polymerase dissociates from template strands
• Some Polymerases add a lot of nucleotides while others add less

Question 8

Describe the proofreading processes in DNA replication.

Answer 8

• Very accurate
• Mistake is 1/10^9-10^10 (every 1000-10000 replications)
• Incorrect pairings are recognized because they don’t form the correct H bonds and don’t fit in active site
• Errors are corrected by exonuclease activity

Question 9

What is PCR?

Answer 9

• Used to selectively amplify DNA sequence in a test tube
• Target DNA, Complimentary primers on either side of target, Nucleotides, and Thermostable DNA polymerase

Question 10

What happens in PCR?

Answer 10

Melting: 94-95C

Annealing: Cool separated strands and primers anneal

Elongation: Polymerase extends primers in 5’ to 3’ (72C)

Repeat 25-30 times in 30 min

Question 11

What is DNA sequencing?

Answer 11

• A template strand has ddNTPs added and can be identified by gel electrophoresis

Question 12

What is Plasmid based gene cloning?

Answer 12

• Self replicating circular DNA
• Circular DNA separated from bacterial genomic DNA
• Replicate autonomously
• Allow DNA cloning up to 15000 BP

Question 13

What are the blotting techniques?

Answer 13

Southern: Used to detect specific DNA sequences with restriction enzymes

Northern: Used to detect and analyze RNA molecules by sized based separation and hybridizes with labeled DNA probes

Western: A method for identifying specific proteins by electrophoresis then membrane transfer using antibodies

Question 14

Design a forward and reverse primer for the following sequence.

Answer 14

Question 15

Describe how the Sanger technique is made and interpreted?

Answer 15

• ddNTPs are added one at a time and radioactively labeled
• Numbers correspond with the sequence of the complimentary strand

Question 16

Describe the two different restrictions of endonucleases.

Answer 16

• Cleave DNA phosphodiester at specific sequences
• In bacteria
• Staggered cuts
• Straight cuts

Question 17

Describe the differences in bacteria and eukaryote genes.

Answer 17

Bacteria:
- Circular DNA
- No exons

Eukaryotic:
- Exon and introns
- double helix DNA

Question 18

What is cDNA?

Answer 18

• When mRNA is used to synthesize a DNA strand via reverse transcriptase

Question 19

What is a cloning vector and how is it used?

Answer 19

• Plasmids are used for sequences up to 15000 BP
• NAC and YAC used for segments up to 300000 BP

Question 20

Describe selection based on antibiotic resistance

Answer 20

• Vector + insert are ligated which causes a transformation which can grow on antibiotic media

Question 21

What are expression plasmids?

Answer 21

• Contain sequences that allow transcription and translated of the inserted gene
• Promoter: where RNA Poly binds
• Operator: allows regulation
• Ribosome binding site
• Transcription termination sequence

Question 22

Describe the structure and function of DNA Polymerase III.

Answer 22

• 2 core domains linked by clamp loader complex
• Interaction with dimer beta subunit increases processivity to greater than 500,000 bp

Question 23

What is a holoenzyme?

Answer 23

• The core, clamp loader, and beta sliding clamp

Question 24

What are the three phases of replication?

Answer 24

• Initiation
• Elongation
• Termination

Question 25

Where does prokaryotic replication initiated?

Answer 25

oriC

Question 26

Outline the important steps of initiation in replication as well as the enzymes involved.

Answer 26

Goal: - Open helix - Form pre-priming complex Steps: - DnaA bind to oriC which unwind helix - DnaB-DnaC bind to unwound DNA - SSBs bind when DnaC dissociates - Primase associates with DnaB (helicase) to make **primasome** to make primers - Fork expands as DnaA dissociates - Pol III binds to fork

Question 27

Outline the important steps of elongation in replication as well as the enzymes involved.

Answer 27

- Parent DNA unwound by helicase - Topological stress relieved by gyrase - SSbs bind separated strands - Primase generates primers at the origin - Deoxynucleotides are added to primer by DNA Pol III - Leading needs one primer while laging needs many

Question 28

How are both strands of DNA replicated by the same complex while both going in a 5' to 3' direction?

Answer 28

- The lagging strand loops around to coordinate simultaneous synthesis in opposite directions

Question 29

What enzymes are involved in the replication fork?

Answer 29

Question 30

Outline the important steps of termination in replication as well as the enzymes involved.

Answer 30

- Replication forks will meet at region with multiple Ter sequences - Ter is where Tus can bind which arrests replication fork -When a fork reaches the non permissible region of the Tus protein (red) it cannot proceed so the forks meet in the middle - In the end the rings of DNA are interlocked until they are unlinked by Gyrase and Topoisomerase II

Question 31

Explain what happens when helicase encounters the red region of the Tus-Ter complex.

Answer 31

Question 32

What role does methylation play in DNA?

Answer 32

- Methylation can regulate the replication of DNA by inhibiting replication initiation

Question 33

What are Okazaki fragments and why are they necessary?

Answer 33

- They are segments of DNA that have not been ligated and they are necessary to allow for synthesis of the lagging strand

Question 34

What three things can DNA Pol I do in E.Coli

Answer 34

5' to 3' polymerase activity 3' to 5' exonuclease activity (proofreading) 5' to 3' exonuclease activity (nick translation)

Question 35

What is the 5' to 3' exonuclease (Nick translation) activity of DNA Pol I? What role does it play in DNA replication?

Answer 35

- When a phosphodiester bond is broken, an exonuclease will remove nucleic acids and add dNTPs using DNA Pol I

Question 36

How does the replicated DNA in bacteria get decatenated?

Answer 36

- Gyrase and Type II topoisomerase