Chapter 20: DNA Replication Flashcards
What is the building block of DNA and RNA?
Nucleotides consisting of nitrogenous base, ribose/deoxyribose sugar, and Phosphoryl group
Which bases are purines and which are pyrimidines? What are the key differences?
Purines: A and G
- Double rings
Pyrimidines C,T,U
- Single rings
- AT form 2 H bonds, CG forms 3 H bonds
What is DNA replication?
- The process of creating copies of DNA
- Semi-conservative
- Begins at origin, proceeds bidirectionally
- Synthesized in a 5’ ->3’ direction (semi-discontinuous)
How was DNA tested to be semiconservative?
Meselson-Stahl
- DNA was grown in a N15 medium, transfered to a N14 and the new DNA had a N14 and N15 strand
Compare a template and a primer.
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
Describe the polymerase mechanism of DNA polymerase.
- 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
What is Processivity?
- The average number of nucleotides added before a polymerase dissociates from template strands
- Some Polymerases add a lot of nucleotides while others add less
Describe the proofreading processes in DNA replication.
- 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
What is PCR?
- Used to selectively amplify DNA sequence in a test tube
- Target DNA, Complimentary primers on either side of target, Nucleotides, and Thermostable DNA polymerase
What happens in PCR?
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
What is DNA sequencing?
- A template strand has ddNTPs added and can be identified by gel electrophoresis
What is Plasmid based gene cloning?
- Self replicating circular DNA
- Circular DNA separated from bacterial genomic DNA
- Replicate autonomously
- Allow DNA cloning up to 15000 BP
What are the blotting techniques?
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
Design a forward and reverse primer for the following sequence.
Describe how the Sanger technique is made and interpreted?
- ddNTPs are added one at a time and radioactively labeled
- Numbers correspond with the sequence of the complimentary strand
Describe the two different restrictions of endonucleases.
- Cleave DNA phosphodiester at specific sequences
- In bacteria
- Staggered cuts
- Straight cuts
Describe the differences in bacteria and eukaryote genes.
Bacteria:
- Circular DNA
- No exons
Eukaryotic:
- Exon and introns
- double helix DNA
What is cDNA?
- When mRNA is used to synthesize a DNA strand via reverse transcriptase
What is a cloning vector and how is it used?
- Plasmids are used for sequences up to 15000 BP
- NAC and YAC used for segments up to 300000 BP
Describe selection based on antibiotic resistance
- Vector + insert are ligated which causes a transformation which can grow on antibiotic media
What are expression plasmids?
- 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
Describe the structure and function of DNA Polymerase III.
- 2 core domains linked by clamp loader complex
- Interaction with dimer beta subunit increases processivity to greater than 500,000 bp
What is a holoenzyme?
- The core, clamp loader, and beta sliding clamp
What are the three phases of replication?
- Initiation
- Elongation
- Termination
Where does prokaryotic replication initiated?
oriC
Outline the important steps of initiation in replication as well as the enzymes involved.
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
Outline the important steps of elongation in replication as well as the enzymes involved.
- 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
How are both strands of DNA replicated by the same complex while both going in a 5’ to 3’ direction?
- The lagging strand loops around to coordinate simultaneous synthesis in opposite directions
What enzymes are involved in the replication fork?
Outline the important steps of termination in replication as well as the enzymes involved.
- 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
Explain what happens when helicase encounters the red region of the Tus-Ter complex.
What role does methylation play in DNA?
- Methylation can regulate the replication of DNA by inhibiting replication initiation
What are Okazaki fragments and why are they necessary?
- They are segments of DNA that have not been ligated and they are necessary to allow for synthesis of the lagging strand
What three things can DNA Pol I do in E.Coli
5’ to 3’ polymerase activity
3’ to 5’ exonuclease activity (proofreading)
5’ to 3’ exonuclease activity (nick translation)
What is the 5’ to 3’ exonuclease (Nick translation) activity of DNA Pol I? What role does it play in DNA replication?
- When a phosphodiester bond is broken, an exonuclease will remove nucleic acids and add dNTPs using DNA Pol I
How does the replicated DNA in bacteria get decatenated?
- Gyrase and Type II topoisomerase
