DNA Replication

DNA Replication: process of copying the genome w/in a cell

  • creates 2 identical DNA molecules, each with 2 complementary strands

  • Purpose: when cell division happens, each cell has their own copy of DNA

Meselson and Stahl: designed an experiment to determine how DNA is replicated

  • demonstrated that it was semi-conservative: After DNA replication, both of the double stranded DNA molecules have one strand of the original DNA & one strand of newly synthesized DNA

  • DNA separates and each of the single strands serve as a template for replication process

HOW DID THEY LABEL THE OLD AND NEW DNA????

  • Conclusion: The complementary base pairing ensures that each new DNA molecule is identical

Initiation of DNA Replication:

  • Helicase binds to the origin of replication (enzyme)

    • place on the DNA molecule where replication will begin

  • Helicase begins to unzip the double helix by breaking the hydrogen bonds between the bases

  • This creates a replication fork: the region where the original DNA double helix splits into two strands

    • y-shaped region where you have the double stranded DNA that hasn’t been unzipped & the two strand that have already been separated

  • Single Strand Binding Proteins: bind to the single stranded DNA to keep the strands separate by preventing hydrogen bonds from reforming

    • the single strand DNA portions really want to come back together and reform hydrogen bonds

  • The process of unzipping the double helix creates supercoils and tension ahead of the replication fork that could damage the DNA

  • Gyrase/topoisomerase: enzyme that moves in front of Helicase to relieve the tension and prevent supercoiling

Synthesizing the Complementary Strand:

  • DNA Polymerase III: the enxyme that will read the template and build the complementary strand

    • it can only add new nucleotides to an existing strand at the 3’ end —> 3’ Sticky End

    • DNA Pol III builds the new strand in the 5’ —> 3’ direction

    • has to build in that direction bc it is an enzyme with an active site that is shaped so that it can only build in that direction

    • NEEDS AN EXISTING 3’ END

  • Primase: creates an existing strand for DNA Pol III to add to the 3’ end

  • It adds a primer made of a short sequence of RNA nucleotides to get DNA Pol III started

    • Once the primer is laid down, DNA Pol III can attach to the new strand and begin synthesizing the complementary strand to the template

  • DNA Pol III reads the template strand to figure out which base needs to be added to the growing complementary strand

    • antiparallel strands

DNA Proofreading:

  • DNA Pol III can proofread the newly formed DNA strand as it is being built

  • If a mistake is made, the mismatched base will be removed and replaced with the correct one

  • DNA Pol III is not 100% accurate with the DNA replication process and proofreading —> Mistakes are still made

    • mistakes are mutations in the DNA which creates variation for Natural Selection to act upon

Removing Primers:

  • *Remember: the primers are made of RNA nucleotides (ribose sugars and Uracils)

  • DNA Pol I: removes the RNA nucleotides and replaces them with the correct DNA nucleotides

    • we don’t want RNA in the middle of our DNA

Directionality in the Replication Fork:

  • *Remember: the DNA strands are antiparallel

  • when helicase unzips the double helix, one strand rund 5’ —> 3’ and the other runs 3’ —> 5’

  • DNA Pol III can only run 5’ —> 3’, so replication does not proceed the same way on both strands

  • Leading Strand: the strand that DNA Pol III can synthesize continuously following the same direction as helicase

  • Lagging Strand: the strand that is synthesized discontinuously AWAY from the replication fork —> works in small chunks

Replication of the Leading Strand: only 1 primer is required to start replication on the leading strand

  • Once the primer is created, DNA Pol III follows the direction of helicase until the whole molecule has been unzipped

Replication of the Lagging Strand: replicated in sections with DNA Pol III moving AWAY from the replication fork

  • These fragments are called Okazaki Fragments

  • each fragment needs its own primer

  • replicates AS the strands unzip —> DNA Pol III keeps looping back to replicate as more is unzipped

Lagging Strand Enzymes:

  • More primase activity —> 1 primer for each Okazaki fragment

  • More DNA Polymerase I activity —> more primers to remove and replace with the correct DNA

  • After the primers have been replaced, the Okazaki Fragments are connected together

  • Ligase: enzyme that catalyzes the formation of phosphodiester bonds between the Okazaki Fragments forming a continuous strand

Applications of DNA Replication:

  • Polymerase Chain Reaction (PCR): used to amplify small fragments of DNA —> create millions/billions of copies

    • *essentially DNA replication in a test tube on steroids

  • PCR Set Up: target sample of DNA is put into a test tube along with:

    • Buffer solution

    • many free DNA Nucleotides

    • Primers for your target sample

    • Taq Polymerase

Taq Polymerase: a DNA polymerase enzyme that is heat stable

  • originally found in a prokaryote that lives in hot springs (thermophile/Archaebacteria)

  • WHAT domain/kingdom is this prokaryote from???

  • able to function at high temps

PCR Steps:

  1. Denaturation: DNA is heated to about 98 degrees C to break the hydrogen bonds between the strands

    • no need for helicase bc of this step

    • denaturing the DNA, not enzymes

  2. Annealing: sample is cooled to 60 degrees C, which allows primers to bind to complementary DNA

    • no need for Primase

  3. Extension: at temps around 72 degrees C, Taq Polymerase replicated DNA

    • DNA Pol III can’t be used bc it would denature in the Denaturation Step

  4. Repeat

What can we do with all of the DNA made in PCR?

Gel Electrophoresis: often done after PCR

  • uses an electrical current to move DNA fragments through a gel network

  • imagine: mesh netting & fragments wiggle their way through the netting

    • smaller fragments can wiggle through faster than larger ones

    • fragments get separated based on size

    • mesh netting = gel network

  • positive charge put at bottom of gel to attract DNA fragments toward it (negative phosphate groups)

  • fragments are separated based on size

  • before you run the gel, it’s treated with restriction enzymes

  • Typically, DNA molecules are too long to travel through a gel, so they must be cut into pieces first

  • Restriction enzymes: cut DNA molecules at very specific sequences

    • AKA restriction endonucleases

    • if there is a mutation at the cut site, the restriction enzymes won’t cut

  • DNA fingerprints can be created based on the banding patterns

  • restriction enzyme cut sites create a unique pattern of bands when a sample is run through a gel “DNA Fingerprint”

  • Single Nucleotide Polymorphisms (SNPs) can change cut sites which causes no enzyme activity at that location, thus changing the banding pattern

    • OR the SNP can create a cut site and allow for enzyme activity that wasn’t previously there

Applications of PCR and Gel Electrophoresis:

  • PCR COVID-19: amplifying the viral nucleic acid to test for the presence of it

  • PCR + Gel Electrophoresis:

    • Paternal testing

    • Forensic investigations