Copying the genetic blueprint DNA replication Copying the genetic blueprint

DNA replication The copying of a cell’s DNA Semiconservative About 3 billion base pairs in the human genome Semiconservative Each strand in the DNA double helix acts as a template for making a new, complementary strand 1 strand2 daughter molecules Each with 1 new & 1 old strand

A:T C:G 5’ 3’ 5’ 3’ DNA Replication = adenine = cytosine = guanine = thymine A:T C:G 3’ 5’

5’ 3’ Step 1…an enzyme breaks the hydrogen bonds between the nitrogen bases and the strand splits 3’ 5’

5’ 3’ 5’ Step 2…each strand of DNA is a template for building a new, complementary strand 3’ 3’ 5’ 3’ 5’

5’ 5’ 3’ 3’ Step 3…completion of the process results in two identical DNA double helices, each with one new and one old strand 3’ 5’ 3’ 5’

DNA replication Basically, that’s it!! HOWEVER…cells need to do this very quickly and accurately What do they need? Enzymes & proteins

DNA polymerase Responsible for synthesizing (putting together) the DNA There are several different DNA polymerases (5 in humans) Add complementary nucleotides one at a time to the growing DNA chain Order based on the template (old DNA strand)

DNA polymerase They always need a template Can’t start out a strand from scratch Require a pre-existing, short length of nucleotides called a primer Can only add nucleotides to the 3’ end of the chain (5’  3’) Proofread their work

DNA polymerase

DNA polymerase Addition of nucleotides requires ENERGY Comes from the nucleotides 3 attached phosphates (like ATP) When the bond is broken, energy is released to form the bond between the nucleotide and the DNA chain

Nucleoside triphosphate (=nucleotide)

How to start??? Origin of replication Replication always starts here Humans can have up to 100,000

Replicaton forks

Helicase & SSB’s 1st replication enzyme at origin of replication Move the replication forks forward by unwinding the DNA (breaking the H-bonds) Single-stranded binding proteins coat the DNA strand to prevent re-annealing

Primase Enzyme Makes an RNA primer Provides a 3’ end for DNA polymerase to add to Typically 5-10 nucleotides long Primes DNA synthesis (gets it started)

DNA polymerase Once the primer is in place: DNA polymerase extends it Adds nucleotides one at a time Can only extend in the 5’ to 3’ direction Leading and lagging strands

Leading & Lagging strands

Leading strand Leading strand is the easy one! 5’ to 3’ Made continuously DNA polymerase moving in the same direction Can be extended from one primer

Leading & Lagging strands

Lagging strand The tricky strand Moves 3’ to 5’ Made in short fragments Okazaki fragments Requires a new primer for each of the short Okazaki fragments

Leading & Lagging strands

Maintenance & Cleanup Sliding clamp Ring-shaped enzyme Keeps DNA polymerase in place Keeps DNA pol of lagging strand from floating off when it re-starts at a new Okazaki fragment

Maintenance & Cleanup Topoisomerase Prevents the double helix ahead of the replication fork from getting too tightly wound and the DNA opens up Makes temporary nicks in the helix to release tension then seals the nicks

Quiz!! Helicase… Opens up the DNA at the replication fork

Quiz!! Single-stranded binding proteins… Coat the DNA strand to prevent re-annealing

Quiz!! Topoisomerase… Works ahead of the replication fork to prevent supercoiling

Quiz!! Primase… Synthesizes RNA primers complementary to the DNA strand; allows DNA polymerase to add on to the strand

Quiz!! DNA polymerase… Extends the primers, adding to the 3’ end

Quiz!! DNA ligase… Seals the gaps between the Okazaki fragments