DNA Replication
• during cell division in eukaryotic cells, the replicated genetic material is divided equally between two daughter cells.
• it is important that each cell gets an exact copy of the parent cell’s DNA.
• in replicating, the DNA double helix unwinds (H-bonds between the strands are broken) and each single strand acts as a template for a new strand.
The Big Picture
Semi-Conservative Replication
• DNA replicates semi-conservatively (each daughter strand contains 1 parent strand and 1 new strand).
• much of our understanding of the process has come from studying prokaryotic systems (like E. coli).
Starting Replication
• replication begins when proteins -DNA helicase, DNA gyrase and single stranded binding proteins) bind to a certain site on the DNA called the replication origin.
Replication Bubble
• the circular DNA in prokaryotes will have 1 replication origin, while in eukaryotes, there are may be several of these along a given stretch of DNA.
DNAP III
• replication of the DNA molecule proceeds in a 5’ to 3’ direction along each strand, thus, in opposite directions.
• nucleotides are added by an enzyme DNA Polymerase III, which adds dNTPs (deoxyribonucleoside triphosphates to a free 3’ end.
Adding Nucleotides
• DNA polymerase III needs a free 3’ end to begin, so another enzyme, primase lays down a 10-60 base pair stretch of RNA primer to allow DNAP III to start.
Adding dNTPs
• note that the dNTPs contain 3 phosphates, while the nucleotides only contain 1.
• DNAP III breaks the bond between phosphates and uses the energy released to add the base to the growing strand.
Leading vs. Lagging
• So, one strand (the leading strand) is synthesized continuously in the 5’-3’ direction, while the other (the lagging strand) is synthesized discontinuously in the 5’-3’ direction.
• lagging strand synthesis works by having RNA primers repeatedly added as the replication fork moves along.
Okazaki Fragments• DNAP III forms small stretches
(100-200 base pairs long in eukaryotes) of new sequence called Okazaki fragments in the 5’-3’ direction.
• DNA Polymerase I removes the RNA primers from both the leading and lagging strands and replaces them with the correct dNTPs.
• the Okazaki fragments are joined together by another enzyme DNA ligase by creating a phosphodiester bond.
• as the new strands are made, two double stranded DNA molecules automatically re-form the double helix.
Quality Control
• because it is so important to ensure that an exact copy of the parent DNA strand is made, both DNAP I and III have a proofreading capability built in.
• both enzymes have exonuclease activity, that is they can remove a nucleotide added in error and replace it with the correct one.
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