8 - DNA Replication Flashcards
(13 cards)
How is bacterial DNA compacted
Looping and supercoiling
How is eukaryotic DNA organised
Tightly coiled and packed together
Error rate of DNA replication in eukaryotic cells
1 in 10^9 chance
Two prediction in Watson-Crick DNA model
- DNA strands are held together by hydrogen bonds between complementary base pairs
A-T - 2 H bonds
C-G - 3 H bonds
- to make this work, the strands must be anti-parallel - Each strands is therefore complementary to the other
- so each DNA strand can act as a template for DNA replication
- so DNA replication should be semi-conservative
3 models for testing DNA replication
- Semi-conservative - one parental and one new strand in DNA molecule
- Conservative - parent helix conserved, daughter helix completely new
- Dispersive - parent helix broken into fragments, dispersed, copies and then assembled into two new helices
- new and old DNA completely dispersed
Results of Meselson and Stahl experiment in 1958
- used CsCl equilibrium density gradient centrifugation to separate molecules on basis of their densities
- found 3 different forms of DNA strands after multiple replications and generations:
- 14N/14N light DNA
- 14N/15N hybrid DNA
- 15N/15N heavy DNA
- showed that DNA replication is semi-conservative
Arthur Kornberg DNA polymerase experiment 1957 - info
- he wanted to work out procedure and conditions required for DNA replication
Procedure: - added multiple chemicals to E. Coli protein extracts
- template DNA required to provide something to copy
- deoxynucleotide triphosphtaes (dNTPs, dATP, etc.) added
- co factor Mg2+ added
- ATP added as energy source
- Primer added - small piece of DNA with free 3 OH group needed
Where does energy for DNA replication come from
- what does this produce
2 phosphate groups removed from phosphate backbone of DNA to release chemical energy from bond
- produces pyrophosphate
- free nucleotide is joined to new DNA strand
DNA replication mechanism
DNA polymerase editing in DNA replication info
- wrong nucleotide can be incorporated at a low frequency
- this leaves an unpaired 3’ OH- end that blocks further elongation by DNA polymerase
- DNA polymerase 3’ —> 5’ exonuclease activity removes the mismatch to leave a base paired 3’ OH end
- DNA replication can now resume
Conclusion of DNA replication by 1960
- DNA strands are anti-parallel
- 5-3 and 3-5
- Watson-Crick complementary base pairing is correct
- DNA replication is semi-conservative
- new DNA made in the 5-3 direction and is mediated by polymerase enzymes that can edit error
- polymerases however, require priming
- DNA polymerase has a proof-reading 3’ to 5’ exonuclease activity
- DNA synthesis is semi-continuous, leading and lagging strand
what are primers and functions
short, single-stranded DNA sequences that are used as a starting point for DNA synthesis in PCR and DNA replication
why are primers needed for DNA replication and polymerase
DNA polymerase can only build nucleotides onto an existing DNA strand
