DNA Replication
Replication is semiconservative
DNA polymerase
- can only add nucleotides to 3’ end
o Fig. 6.17 about how reaction works and where energy comes from
- needs a primer
o Primer can be DNA or RNA (in chromosomal replication it is RNA)
Replication can be thought of in two separate stages
- initiation
- elongation
Initiation
Replication begins at a single point (an origin of replication) and proceeds in both directions (bidirectional replication)
- E. coli (and many bacteria) - one origin of replication on a circular chromosome
o oriC is chromosomal origin
o 240bp sequence (can see similar sequences in most bacterial genomes
o Initiator protein binds to this sequence (actually several copies of protein bind) (can also see similar proteins to this in other bacterial genomes)
§ This “unwinds” the DNA – local denaturation to make replication bubble
§ Results is two replication forks
- Humans and many eukaryotes - many origins of replication
o not all origins fire at the same time, so early, some late
o many sequences can act as origins, so they are impossible to pick out accurately by computer
o an initiator protein binds to origin
Elongation
- first a primer is laid down by the enzyme primase
o primer is a short (15nt or so) RNA
- then DNA polymerase attaches to 3’ end of primer and starts adding DNA
o In E. coli this is DNA polymerase III
o In humans still not clear – whether one or two collaborate
- DNA is synthesized quite differently on the two different strands
o One is synthesized continuously (leading strand)
o One is synthesized discontinuously (lagging strand)
§ lagging strand synthesized as a bunch of Okazaki fragments, each has RNA at 5’ end
§ Okazaki fragments
· 1000 bp in prokaryotes
· 100 or so in eukaryotes
- Enzyme called helicase travels ahead of the DNA polymerases, denaturing (unwinding) the DNA ahead
o Protein called single strand binding protein (SSB) binds the single stranded DNA transiently until DNA polymerase comes along, protecting it
- Need to degrade RNA (in primers) and replace with DNA
o In E. coli it is DNA polymerase I that does this
o Eukaryotes it is a combination of enzymes including a DNA polymerase
- Final step – DNA ligase makes the final phosphodiester bond after the RNA has been degraded and filled in with DNA
Topoisomerases
- topoisomerases to relax tension built up when helicase unwinds DNA
Accuracy
- overall error rate is about 1 error every 1010 nt
- E.coli genome is 4.7 X 106 bp, so 1 error every 1000-10000 replications
- Human genome is 6 X 109 bp, about one error every replication
PCR
- Polymerase chain reaction
- A technique
- Invented in 1983 by Kary Mullis (Nobel prize 1993)
- A way to make many copies of a nucleic acid starting from just a few (or even one!) (amplification)
- Add to a tube
o the DNA you want to copy (template)
o short single stranded oligonucleotides that are complementary to sequences that flank the region you want to amplify
o dNTPs
o DNA polymerase that is stable at high temps
§ Isolated from bacterium in Yellowstone Park hot springs, Thermus aquaticus
· Taq polymerase
- Repeating cycles of three steps
o Denaturation
§ Heat up to separate DNA strands (usually 95oC or so)
§ Breaks H-bonds between strands, so strands separate
o Annealing
§ Cool to allow the oligonucleotides to basepair with the template (40-50oC, but this varies tremendously from application to application)
o Extension
§ Heat to optimal temp of DNA polymerase, it will extend 3’ end as DNA polymerases always do and make DNA (72oC)
- At third cycle get first 2 pieces of dsDNA that is exactly what you want amplified
- 4th cycle have 4
- 5th cycle have 8
- 10th cycle have 256
- 20th cycle have 262,144
- 30th cycle have 268,435,456