Molecular Biology Lecture 5
DNA Replication Semiconservative Daughter DNA is a double helix with 1 parent strand and 1 new strand Found that 1 s...
DNA Replication Semiconservative Daughter DNA is a double helix with 1 parent strand and 1 new strand Found that 1 strand serves as the template for new strand
DNA Replication
5’
Origins of replication 1. Replication Forks: hundreds of Y-shaped regions of replicating DNA molecules where new strands are growing.
3’
Parental DNA Molecule
Replication Fork
3’ 5’
DNA Replication
Origins of replication 2. Replication Bubbles: a. Hundreds of replicating bubbles (Eukaryotes). b. Single replication fork (bacteria).
Bubbles
Bubbles
DNA Replication
Strand Separation: 1. Helicase: enzyme which catalyze the unwinding and separation (breaking HBonds) of the parental double helix. 2. Single-Strand Binding Proteins: proteins which attach and help keep the separated strands apart.
DNA Replication
Strand Separation: 3. Topoisomerase: enzyme which relieves stress on the DNA molecule by allowing free rotation around a single strand. Enzyme
DNA
Enzyme
DNA Replication
Priming: 1. RNA primers: before new DNA strands can form, there must be small pre-existing primers (RNA) present to start the addition of new nucleotides (DNA Polymerase). 2. Primase: enzyme that polymerizes (synthesizes) the RNA Primer.
DNA Replication
Synthesis of the new DNA Strands: 1. DNA Polymerase: with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ direction.
5’
3’
Nucleotide
DNA Polymerase
RNA Primer
5’
DNA Replication 2. Leading Strand: synthesized as a single polymer in the 5’ to 3’ direction.
5’
3’ 5’ Nucleotides
DNA Polymerase
RNA Primer
DNA Replication 3. Lagging Strand: also synthesized in the 5’ to 3’ direction, but discontinuously against overall direction of replication. Leading Strand
5’ 3’
DNA Polymerase
3’ 5’
RNA Primer
5’
3’
3’
5’
Lagging Strand
DNA Replication 4. Okazaki Fragments: series of short segments on the lagging strand.
DNA Polymerase
Okazaki Fragment RNA Primer
5’
3’ 5’
3’ Lagging Strand
DNA Replication 5. DNA ligase: a linking enzyme that catalyzes the formation of a covalent bond from the 3’ to 5’ end of joining stands. Example: joining two Okazaki fragments together. DNA ligase 5’
Okazaki Fragment 1
3’
Okazaki Fragment 2
3’
5’ Lagging Strand
DNA Template
Each strand of the parent DNA is used as a template to make the new daughter strand DNA replication makes 2 new complete double helices each with 1 old and 1 new strand
Replication of DNA
Self compatible nature of DNA allows the two molecules of parental DNA to serve as template for replication Complex and multistep processes involves various enzymes After this lecture we will be able to Distinguish between leading and lagging strands Understand the function of DNA replication proteins How antiparallel stands are copied simultaneously
Process of DNA replication
Helicases are hexameric proteins in the form of a ring and it couple hydolysis to the strand separation Junction: replication fork SSB binding occurs to keep the strands separate As the DNA unwinds the twist number decreases and writhe number increases and causes the DNA to become positively supercoiled and removed by topoisomerases
Process of DNA replication conti…
RNA primer is required in the start to start the process of replication Primase are activated by other proteins like DNA helicase DNA Primase adds up an RNA primer Synthesis of DNA is catalyzed by DNA polymerase enzyme DNA Pol. Add dNTPS at 3’ end of polynucleotide chain. 1 Sec for attachment Capable of adding upto 1000 bp/ sec Processivity ? DNA sliding clamps
Process of DNA replication conti..
Simple addition of nucleotides along one strand, as expected Called
the leading strand
DNA polymerase reads 3’ 5’ along the leading strand from the RNA primer Synthesis proceeds 5’ 3’ with respect to the new daughter strand Remember how the nucleotides are added!!!!! 5’ 3’
Process of DNA replication conti.. Other daughter strand is also synthesized 5’3’ because that is only way that DNA can be assembled However the template is also being read 5’3’
Compensate
for this by feeding the DNA strand through the polymerase, and primers and make many short segments (Okazaki fragments) that are later joined (ligated) together
Called the lagging strand
Process of DNA replication cont..
Rnase H Degrades the RNA that is base paired with DNA Primer leaves the gap DNA pol fill the gaps Nicks are filled by DNA ligases In bacteria DNA polymerases are often linked in holoenzyme Holoenzyme?
A multi proteins complex in which the core enzyme activity (Such as DNA pol. ) is assosiated with additional components that enhances the function
Mistakes during Replication
Base pairing rules must be maintained
Mistake = genome mutation, may have consequence on daughter cells
Only correct pairings fit in the polymerase active site If wrong nucleotide is included
Polymerase uses its proofreading ability to cleave the phosphodiester bond of improper nucleotide
Activity 3’ 5’
And then adds correct nucleotide and proceeds down the chain again in the 5’ 3’ direction
