Lecture 2 - DNA Replication

What must DNA do before cell division?

DNA must be replicated to make 2 identical copies for the daughter cells

Origin of replication

Specific sites (sequences) where DNA replication starts

Replication bubble

expanding area of replicated DNA

Replication fork

site of active replication, two per replication bubble

DNA polymerase can...

add new nucleotides to the 3' end of a DNA strand

Limitations of DNA Polymerase

1. cannot unwind double-stranded DNA

2. cannot start a chain, can only add a nucleotide to an existing nucleotide strand

3. can only add nucleotides to 3' end

4. cannot link existing DNA chains together

DNA Helicase

- found in replication forks

- unwinds double-stranded DNA

- BUT overwinds the DNA in front of it (requiring Topoisomerase)

Primase

builds short RNA strands called primers that allow DNA Polymerase to build a DNA strand

Leading strand

- continuous

- synthesize towards the fork (5' -> 3')

*think leading so will start at the higher prime (5') end+

Lagging strand

- discontinuous (in Okazaki fragments)

- synthesize away from the fork (3' -> 5')

- fragments closer to the origin of replication are built first

Sliding clamp

Allows DNA Polymerase to keep going without falling off

Single stranded binding proteins (SSBPs)

prevent lagging strand from folding on itself and blocking replication

Multiple DNA polymerase are used in DNA replication, what are they?

DNA polymerase 3 and 1

Exonucelase

cuts nucleotides off end of nucleic acid strand

DNA polymerase 3

- main DNA polymerase (builds the RNA strands)

- 5' -> 3' polymerase

- 3' -> 5' exonuclease (for proofreading)

DNA polymerase 1

- specialized DNA polymerase, removes the polymerase so strands can build where it once was

- 5' -> 3' polymerase

- 3' -> 5' exonuclease (for proofreading)

- 5' -> 3' exonuclease (for replacing RNA primers)

DNA Ligase

connects adjacent strands of DNA together to combine fragments to form one continuous strand

Nucleotide triphosphate and its purpose

- new nucleotides enter as nucleotide triphosphates (3 phosphates connected to the nucleotide, ex. ATP)

- two out of the three phosphates are loss which releases energy to power the connection of the nucleotide to the strand

Why are nucleotides added to 3' end?

Allows for proofreading and removal of incorrect nucleotides

Telomeres

- non-protein coding repetitive sequence found at ends of chromosomes

- act as buffer to protect protein coding genes

- shorten w/each replication

- limits replicative potential for cells (ex. cells can replicate forever and forever; prevents cancer but may contribute to aging)

Telomerase

- protein-RNA complex

- because telomeres shorten w/ each replication it means the chromosome itself also shortens w/ each replication

- Telomerase counters this by extending the 3' end of DNA strands

Who discovered telomeres and telomerase?

Elizabeth Blackburn