Lectures 3-5 Flashcards
How did Messelson and Stahl show that DNA replication is by semi conservative mechanisms and what does this mean?
Grew e.coli in 15N a heavy isotope, then switched to 14N a lighter isotope, centrifuged, measured ratio of 15N DNA to 14N DNA
Explain the 6 steps how DNA is replicated in prokaryotes
- Dna A recognizes the OriC and distorts
- Dna B and (helicase) and Dna C open up DNA, unwinds using ATP creating a bidirectional replication fork
- SSBP bind to the strand to prevent formation of hair pins
- (unwinding by Dna b creates positive supercoils) DNA gyrase (type 2 topiosomerase) removes the positive super coils by doubled stranded breaks
- DNA primase (dna G product) synthesizes RNA primers on template providing a free 3’OH
- polyermization by DNA pol III 5’->3’ activity, DNA pol I removes primers with exonuclease and polymerase acitivty, DNA ligase seals nicks
what is the primosome
DNA primase and DNA helicase, initiates okazaki fragments on lagging strand
what is the replisome
complete replication apparatus
primosome, and DNA pol III holoenzyme
lagging stand is looped, both DNA pol III are attached so the one on the lagging strand can catch a ride, new clamps loaded
describe the DNA poly III holoenzyme
catalytic core: alpha has polermerase activity, eplison has 3’->5’ exonuclease acitivty, 0 stimulates proof reading by epsilon
sliding clamp: 2 beta sub units
What are the difference of DNA replication in prokaryotes and eukaryotes?
Pro: done in 40 mins, is conintious, 1 circular chromosome, 1 origin of replication, plicase has 2 catalytic cores
Euks: 9 hours, only in S phase, several pairs of linear chromosomes, multiple orgins of replication, 2 or more polyermases engaged at replication fork, DNA packed in nucleosomes
Describe the miltipule polmerases at the replication fork in eukaryotic DNA replcation
alpha: together with DNA primase, prime and synthesize short Okzaki fragments on lagging, delta: extends the primers on the LAGGING strand, epsilon: replicated the LEADING strand
Descrie how primers are removed in eukarotic DNA replication
Delta and Epsilon lack 5’-3’ exonuclease activity so RNA primers are removed by ribonucleases
What are telomeres
Ends of linear chromosomes in eukaryotes, overhang of 3’ end creates a t-loop, coates in shelterin complexes: TRF 1 and 2,and POT 1
How are telomeres replicated?
DNA pol 1 cannot fill gap at end of chromosome, ends grow shorter with each cell division. Unique enzyme to solve problem, telomerease recognizes the repeast sequence and extends 5’-3’ one at a time, contains own RNA template, compoased of protein and RNA (riboprotein complex), uses reverce trancriptase -> synthesizes DNA using RNA template, DNA polermerases syntehsizes the complementary strand
Explain how DNA is replicated in prokaryotes
- Dna A recognizes the OriC and distorts
- Dna B and (helicase) and Dna C open up DNA, unwinds using ATP creating a bidirectional replication fork
- SSBP bind to the strand to prevent formation of hair pins
- (unwinding by Dna b creates positive supercoils) DNA gyrase (type 2 topiosomerase) removes the positive super coils by doubled stranded breaks
- DNA primase (dna G product) synthesizes RNA primers on template providing a free 3’OH
- polyermization by DNA pol III 5’->3’ activity
what is the primosome
DNA primase and DNA helicase
Describe the miltipule polmerases at the replication fork in eukaryotic DNA replcation
alpha: together with DNA primase, prime and synthesize short Okzaki fragments on lagging, delta: extends the primers on the LAGGING strand, epsilon: replicated the LEADING strand
Descrie how primers are removed in eukarotic DNA replication
Delta and Epsilon lack 5’-3’ exonuclease activity so RNA primers are removed by ribonucleases
Describe DNA replication in Prokaryotes, what are the basic requirements and why are they important
Bidirectional and begins at single initiation site OriC, DNA synthesis requires, DNA pol III, template strand, a primer and all 4 dNTPs
