DNA Replication Flashcards
Aicardi-Goutieres syndrome
Cerebrospinal fluid has elevated white cells (lymphocytes) and high levels of interferon-alpha (INF) activity
Atrophy in frontal and temporal regions of brain
Followed by loss of almost all motor skills
Initiation- prokaryotic replication
One start site for DNA replication –> initiation site
In ecoli: oriC
Initiator protein + ATP + histone like proteins attach to oriC causing DNA to wrap around the complex creating negative superhelical tension in the adjacent DNA
Results in the opening of the double helix at 3 x 13 AT rich repeats left to the protein complex
Further opening the DNA creates a replication bubble
oriC
oriC has 4 sequences 9 bp in length that are recognized by the initiator protein
Adjacent to oriC are 3 AT rich repeats
Why are AT rich repeats important?
Provides a site for DNA to open easily (more unstable than GC rich DNA)
How many replication forks per replication bubble?
2
What are the limits of DNA polymerase?
DNA polymerase cannot initiate DNA synthesis (new strand synthesis)
Requires a 3’ hydroxyl group at the end of a base paired strand to add dNTPs
RNA primers
synthesized 5’ –> 3’ and are laid down antiparallel and complementary to the DNA template strands (both stands of the parent DNA)
Reaction is catalyzed by primase
primosome
primase+ helicase + protein
binds both strands of the replication bubble and makes a short RNA primer
DNA polymerase 1
RNA primer excision
DNA repair
3’ and 5’ exonuclease activity
not the main synthetic enzyme in E coli
Processivity: 3-200
3’ exonuclease activity
when an incorrect base is incorporated the 3’ exonuclease removes it, correct base can be inserted
5’ exonuclease activity
removal of RNA primers in Ecoli
DNA polymerase 3
synthesizing enzyme in replication- replicative chain elongation
processivity- 500,000
functions as a holoenzyme, with a core of 3 polypeptides
Core of DNA polymerase 3
alpha- dimer polymerase
epsilon- dimer 3’ exonuclease for proofreading
beta- dimer that forms sliding clamp around DNA
dimer so that it can replicate both strands of DNA at the same time
beta dimer in pol III
internal diameter of 3.5 (just enough to fit 2 nm diameter for DNA) that has a low affinity in its inner surface to DNA so that the DNA ca slide smoothly along it
Gamma complex of DNA polymerase 3
made up of various holoenzymes that help the beta subunit unload onto the DNA –> clamp loading
Chi and Psi subunits of DNA pol 3
Also bind to the g complex
Chi subunit mediates transition from synthesizing RNA primers to DNA
T complex DNA pol 3
Ensures that the core enzyme is a dimer, allowing both strands of the replication fork to be synthesized simultaneously
also binds to helicase
Lagging strand synthesis
Has to go from 5 –> 3
the fork has to open up BEFORE new synthesis can be initiated
What is the function of the loop?
Allows DNA synthesis to run smoothly and in the same direction for both strands
lagging strand folds to make a small loop, this allows the lagging strand to be in the same orientation as the leading strands and now they can be copied together by the holoenzyme dimers.
What is formed at the end of DNA replication for bacteria?
2 lagging strands and 2 leading strands of new DNA
Termination- prokaryotic replication
termination region is 180 degrees from the oriC (ter region)–> 20 bp segment
when replication fork meets TUS-ter complex, it halts…ensuring that each of the two forks initiated at oriC will travel no farther than ter
Proofreading
DNA Polymerase III can recognize mismatched base pairing (mainly AG and CT)
The epsilon subunit removes the mismatched base pain with its 3’ to 5’ exonuclease activity
The chain is then extended by DNA polymerase III
Nick Translation
DNA polymerase I 5’ to 3’ exonuclease hydrolyses the RNA primers
Simultaneously, the 3’ end of the Okazaki fragment (DNA) is extended by incorporation of deoxyrobonucleotides
DNA pol 1 moves to replace the RNA fragments in the previous Okazaki fragment with DNA, the “nick” keeps moving over one space until the end where ligase seals it together
Final step in termination of DNA synthesis in E coli
type II topoisomerase separates the 2 interlinking strands of the replicated circles and refold the DNA into supercoils
DNA polymerase α
Found in nucleus
Has primase activity –> can make RNA primer in lagging strand replication
Moderate inherent processivity
Moderate processivity with PCNA (like a clamp)
DNA polymerase δ
Found in nucleus Lagging strand synthesis Low inherent processivity High processivity with PCNA 3' exonuclease activity
DNA polymerase ε
Found in nucleus Leading strand synthesis High inherent processivity High processivity with PCNA 3' exonuclease activity
DNA polymerase γ
Found in mitochondria mitochondrial DNA replication High inherent processivity High processivity with PCNA 3' exonuclease activity
*not sensitive to aphidicolin
Sensitivity to aphidicolin (fungal steroid that inhibits replication)
To see which specific polymerases were associated with replication
3 prime structure in replication
Nucleosomes are dissociated ahead of the replication fork, and reassembled on newly synthesized DNA
Okazaki fragments in eukaryotic DNA replication
Size is much smaller than in prokaryotic synthesis
Telomeres
Highly repetitive sequences at the 3’ ends of linear chromosomes
What is added by telomeres?
Protein component enzyme- reverse transcriptase- hTERT (human telomerase reverse transcriptase”
RNA component- hTR (human telomerase RNA)
Template
Why do we need telomeres?
When you come to the last primer in the lagging stranding, polymerase 1 cannot complete gap at the end –> need OH group for DNA pol to work
How does telomerase work?
Telomerase uses RNA component to add repeats
Telomerase repositions itself to add each repeat (moves 3’ –> 5’ on daughter strand)
The daughter strand is synthesized using complementary base pairing by DNA polymerase alpha
This process is repeated ultimately forming the long telomere
3’ overhang at telomere end
A single stranded 3’ overhang left when telomerase moves away
D-loop T-loop
Telomere ends are protects by loops that are stabilized by binding of telomere-binding proteins called TTAGGG repeat binding factors
