DNA Replication Flashcards
What are the four steps of DNA synthesis?
- Initiation
- Priming
- Synthesis
- Termination
How many initiation sites are there in prokaryotes?
1
Is prokaryotic synthesis unidirectional or bidirectional?
bidirectional
Why are AT rich repeats important for initiation?
They are less stable than native DNA and can open to form the replication bubble
What MUST come before DNA polymerase?
RNA primase!!
In what direction is DNA made?
5’ to 3’
What is the function of DNA pol III?
Replicative chain elongation
What is the function of DNA pol I?
Primer excision (nick translation) and DNA repair
Does DNA pol I or III have higher processivity?
Pol III
What does the beta subunit of Pol III do?
A sliding clamp that encircles DNA and holds the polymerase onto the DNA
What does the alpha subunit of Pol III do?
Polymerize DNA
What does the epsilon “ε” subunit of Pol III do?
Proof reading/ 3’ to 5’ exonuclease
How many Pol III complexes will you have?
2! One at each replication fork
On the leading strand DNA is synthesized in the _____ direction as the replication fork and on the lagging strand DNA is synthesized in the _______ direction as the rep fork.
Leading = same
Lagging = opposite
Single strand binding proteins protect open DNA on the _____ strand.
Lagging
What does the gamma complex do in DNA Pol III?
It loads the beta subunit clamp
How are RNA primers removed from the lagging strand?
DNA Polymerase I in a process called Nick Translation
How does Nick Translation work?
DNA Pol I uses 5’ to 3’ exonuclease activity to hydrolyze primers and simultaneously the 3’ end of the Okazaki fragment is extended by incorporation of dNTPs. The nick is then sealed by DNA ligase
What enzyme joins all the okazaki fragments?
DNA ligase
What is the final step of e. coli DNA synthesis?
Type II topoisomerase separates interlinking strands of the replicated circles and refolds the DNA into supercoils
What does DNA polymerase alpha do in eukaryotic DNA?
Makes the RNA primer at both leading and lagging strand
Rnase H is defective in Aicardi-Goutieres syndrome. What is Rnase H responsible for?
Removal of RNA primer during eukaryotic DNA replication
What is the diagnostic feature of Aircardi-Goutieres syndrome?
INF alpha in CSF
What is the electrostatic charge of the inside of the inside of the Pol III B-subunit?
Negative–has a low affinity for DNA and keeps from sticking
What is the origin of replication called in prokaryotes and eukaryotes?
OriC
ARS–autonomous replicating sequences
What binds the origin of replication and initiates replication in prokaryotes and eukaryotes?
dnaA complex
ORC–origin of replication complex
What helicase is used in prokaryote and eukaryote DNA replication?
dnaB/dnaC complex
MCM–minichromosome maintenance proteins
What stabilizes single stranded DNA in prokaryotes and eukaryotes?
SSB’s
RPA–replication protein A
What increases the processivity DNA replication?
B-subunit of Pol III
PCNA-proliferating cell nuclear antigen–of DNA Pol delta 𝛿
What serves as the clamp loader in DNA replication in prokaryotes and eukaryotes?
γ gamma subunit
RFC-replication factor C
What makes the RNA primer in prokaryotes and eukaryotes?
dnaG
DNA Pol α alpha
What removes the RNA primers in prokaryotes and eukaryotes?
DNA Pol I (nick translation)
Rnase H and FEN1 (flap endonuclease)
What eukaryotic polymerase is responsible for synthesizing DNA on the leading strand?
DNA Pol ε epsilon
What eukaryotic polymerase is responsible for synthesizing DNA on the lagging strand?
DNA Pol δ delta
What eukaryotic polymerase is responsible for synthesizing mitochondrial DNA?
DNA Pol γ gamma
What are the two components of telomerase?
hTERT–protein component–reverse transcriptase
hTR–RNA component–serves as template
What type of structure stabilizes the binding of telomere-binding proteins?
D-loop-T-loop
What is the difference between exonucleases and endonucleases?
Exo–cleave at the end of polynucleotide chain
End–cleave in the middle of polynucleotide chain
Describe the structure of DNA polymerase III
Consists of a core enzyme (theta, sigma, alpha, beta, tau) and a gamma-complex clamp loader
