Unit 3: DNA Replication Flashcards
Why is our DNA semi-conservitive?
Each DNA strand serves as a template strand to produce a new complementary one
What do the 2 Mg2+ ions held in Pol do?
1st Mg2+: Deprotonates the primer 3’-OH hydroxly to 3’-O- nucleophile
2nd Mg2+: Binds PPi and facilitates its departure
What are the DNA polymerase reaction steps?
- Synthesis of the RNA primer
- Incorporation of the 1st nucleotide
- Translocation of polymerase
- Incorporation of 2nd nucleotide
How does the DNA polymerase know which dNTP to incorporate?
Binding of a dNTP causes a conformational change of Pol Active site in closed form
What kind of proofreading activity does DNA polymerase have?
A 3’ - 5’ exonuclease to get rid of incorrect nucleotides added
What does Pol III contain?
Pol III core x3
Sliding Clamp x3
Clamp loader
What is the role of the Pol III core?
Add the nucleotides & proofreading
What is the role of the Pol III beta sliding clamp?
Increase speed of replication by not letting polymerase fall off of DNA
What is the role of the clamp loader?
Loads beta sliding clamp onto DNA
What does the DNA helicase do? Where is it located?
DNA “unzipping”
In bacteria: located on lagging strand
In eukaryotes: located on leading strand
What does the Topoisomerase do?
Works ahead of the Helicase to untwist DNA
What does the Primase do?
RNA polymerase that adds RNA primer as the first nucleotides
What does a ligase do?
Seals DNA nicks, “glues the Okazaki fragments together”
What is a nick in DNA?
A place where the phosphdiestar bond is not made between fragments
What occurs during nick translation?
The Pol I exonuclease works 5’ to 3’ to remove RNA primers and replace with DNA
What happens after Pol I is done?
After Pol I, beta clamp stays on to recruit ligase
How is the beta clamp removed?
Delta subunit of clamp loader
What is 1 difference between bacteria and eukaryotic DNA replication structures?
Eukaryotic replisome has 2 DNA polymerases (1 for each strand) as opposed to 3 so it is much slower
How does replication initiation begin in bacteria?
A long bp sequence with repeating bp sites is recognized by DnaA and the DnaA binds to these R-repeats. An A&T rich repeat unwinds the DNA
What happens after the A-T region is destabilized?
DnaC loads helicase onto each strand
What prevents the origin from initiating again?
DAM sites are methylated in old strands, but new strands are not allowing SeqA to bind to the unmethylated strand and prevent DnaA from binding to origin.
How do eukaryotes orchestrate chromosomal replication? What are the steps?
Through cell cycle phases
- # 1 G1: Assembly of pre-replication complexes
- # 2 S: Degradation of preRC
- # 3 S: Replication
- # 4: S-G2-M: no pre-RC assembly = no initiation
