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DNA Replication
2.
3.
Conservative
Dispersive
http://mol-biol4masters.masters.grkraj.org/html/Prokaryotic_DNA_Replication1-Introduction.htm
The E. coli chromosome is circular Preserves the integrity of the circular chromosome DNA replication initiates at a single point and proceeds from one or two replication forks
DNA Synthesis
Requires DNA Polymerase 1. 3 DNA polymerases : I, II, and III - polymerase III : replicates most of the DNA 2. Synthesizes DNA in a 5 to 3 direction
Catalyzes formation of phosphodiester bond between 5 PO4 of deoxyribonucleotide and 3 OH on DNA strand - substrate is deoxynucleotide triphosphate
http://en.wikibooks.org/wiki/Medical_Physiology/Cellular_Physiology/DNA_and_Reproduction
Energy for polymerization comes from cleaving 2 phosphates from deoxyribonucleotide triphosphate
leading strand
lagging strand
Lagging strand is synthesized discontinuously (in pieces: Okazaki fragments) in direction opposite of movement of replication fork
(~150bp in eukaryotes)
Primer Synthesis
Primase synthesizes a primer (10-12 nucleotides long) complementary to DNA template strand
Elongation
DNA polymerase III adds deoxyribonucleotides to the 3 OH end of the primer
Elongation
Fidelity : a measure of polymerase accuracy at incorporating correct deoxynucleotide (E. coli : 1 mismatch in 109 base pairs !)
why is primer made of RNA ? - primers are not always exact matches to template RNA primer can be removed and correct DNA sequence added by pol III
53 exonuclease activity of DNA polymerase I removes primer 53 polymerase activity of DNA polymerase I fills in the gap with DNA
Ligation
DNA Ligase seals up nicks left after DNA polymerase has filled in the gaps
ligase : catalyzes formation of a phosphodoester bond from a 5 phosphate and 3 OH
continuous synthesis
discontinuous synthesis
holoenzyme : protein complex with all associated subunits DNA pol III : 10 subunits total - 3 form core enzyme required for activity - subunit : clamp that holds polymerase onto DNA processivity
for overall movement in direction of replication fork : lagging strand must loop through DNA pol III to allow 53 synthesis of Okazaki fragment away from replication fork
polymerase cycling : moving off and on the lagging strand template as Okazaki fragments are completed
3. recruit
clamp
Supercoiling
Topoisomerases can put in or remove supercoils from the DNA
negative : circular DNA winds around itself in the opposite direction as the twist of helix (left handed)
positive : circular DNA winds around itself in the same direction as the twist of helix (right handed)
Topoisomerases
Change the supercoiling of the DNA by increasing or decreasing the linkage number Type I Topoisomerases cut one of the DNA strands, insert unbroken end in opening Type II Topoisomerases cut both DNA strands, insert unbroken double helix through opening i.e., gyrase
Topoisomerases
Required for DNA replication As helix opens during DNA replication, positive supercoils occur in front of the replication fork gyrase removes those supercoils so that DNA replication can proceed
Topoisomerase
2. D loop
Telomerase
RNA + enzyme complex that binds and extends 3 end of linear chromosomes ** ultimate goal : not to increase length, but preserve telomere 1. Telomerase RNA base pairs with single-stranded 3 end of DNA
several times
2.
Telomerase extends telomere by reverse transcription (RNA from telomerase is template) adds GGGGTT (or similar) sequence Telomerase translocates to extended 3 end
3.
- then primase, polymerase, and ligase make DNA strand complementary to the new telomere sequence
Telomerase
1. Telomerase RNA base pairs with DNA
Telomerase
2. Telomere extension occurs reverse transcription
Telomerase
3. Telomerase translocates to extended 3 end
Telomerase
4. Telomerase extends 3 end of telomere reverse transcription
exact size of telomere may fluctuate from one round of DNA replication to the next, but . . . ** genomic information adjacent to telomere is preserved **