Lecture 24 Flashcards
Helicase
Unwinds double helix by breaking H bonds between the complementary nitrogenous base pairs in the rungs between 2 strands
Allows 2 parental template strands to separate from each other
As DNA helicase does its work, it causes DNA double helix to twist more tightly in front of the moving replication fork
DNA polymerase
Proteins in dna synthesis
Add nucleotides only to an existing strand
Opening up of parental DNA doesn’t provide any strands with an exposed 3’ end to which DNA polymerase can attach
A new strand begins with a short chain of RNA (primer) synthesized by the enzyme primase
Topoisomerase
Role is to alleviate additional coiling from helicase
Does this by breaking swiveling and rejoining DNA strands ahead of the moving replication fork
Separated strands are prevented from coming together again behind DNA helicase by single- stranded binding proteins which bind to each separated strand
Primase
Builds a complementary RNA primer consisting of 5-10 nucleotides
Leaves the template and DNA polymerase takes over, extending RNA primer with DNA nucleotides as it synthesizes the new DNA chain
RNA primers are replaced with DNA later in replication
What does primase have to remove
Removes single stranded binding proteins before it can make the primer
Primases make RNA primer along each of the exposed parental template strands
-toward the expanding replication fork beside the leading strand template
-and away from expanding fork beside the lagging strand template
Once primer is completed , primase detaches from the template strand and a DNA synthesizing enzyme called DNA polymerase 3 attaches to the exposed 3’ end of the RNA primer
DNA polymerase III
Removes single stranded binding proteins and begins to build a daughter strand by adding DNA nucleotides to the daughter strand
DNA polymerase 3 extends the daughter strand by attaching DNA nucleotides onto the strand in the 5’ to 3’ direction
New complimentary nucleotides are added to the daughter strand according to the AT/GC rule
Synthesizing the leading strand
Only 1 primer is necessary to start synthesis of the leading strand template
DNA polymerase III once attached to this primer synthesizes the leading strand continuously towards the moving replication fork
Synthesizing lagging strand
Each RNA primer is synthesized in the opposite direction away from the replication fork
DNA polymerase 3 attaches to a newly synthesized primer and synthesizes DNA away from the replication fork
This happens only for a short distance producing a short piece of DNA called an Okazaki fragment
DNA polymerase 3 has to detach from the fragment it’s synthesizing bc it encounters the RNA primer of a previously synthesized Okazaki fragment
So DNA polymerase 3 releases from the fragment it has been synthesizing leaving an Okazaki fragment in its place
As the helicase opens up more strand, additional RNA primers are added and addition Okazaki fragments are added
DNA polymerase 1
Attaches to Okazaki fragment 2 at its 3’ end and begins to remove nucleotides on the RNA primer of the fragment 1 and replaces the RNA with nucleotides with DNA nucleotides to the 5’ to 3’ direction
When DNA polymerase 1 encounters the first DNA nucleotide of fragment 1 the enzyme detaches from fragment 2 and leaves
DNA polymerase 1 leaves a nick in the sugar phosphate backbone of the lagging strand between fragments 2 and 1
Nick
Protein synthesis
A discontinuity in a double stranded dna molecule where there is no phosphodiester bond between adjacent nucleotides of one strand
DNA ligase
Needed to complete the covalent phosphodiester bind that will repair the Nick left by DNA polymerase 1
It then releases and leaves
proteins in DNA synthesis summary
DNA helicase unwinds
Polymerase 3 adds nucleotides towards fork on leading strand
RNA primers are added to lagging strand
Polymerase 3 adds nucleotides away from fork on lagging strand until the RNA primer is reached
Polymerase 1 replaces RNA primer
Leaves a nick that DNA Ligase fixes
Summary of proteins of replication
Helicase
Unwinds DNA helix
Summary of proteins of replication
Single stranded binding proteins
Stabilize single stranded DNA and prevent the 2 strands at the replication fork from reforming double stranded DNA
Topoismerase
Summary of proteins of replication
Avoids twisting of DNA ahead of replication fork (in circular DNA) by cutting DNA turning the DNA on one side of the break in the direction opposite to that of the twisting force and rejoining the 2 strands again
Primase
Summary of proteins of replication
Synthesises RNA primer in the 5’ to 3’ direction to initiate a new DNA strand
DNA polymerase 3
Summary of proteins of replication
Main replication enzyme in E COLI
Extends the RNA primer by adding DNA nucleotides to it
Summary of proteins of replication
DNA polymerase 1
E. coli enzyme that uses its 5 to 3’ exonuclease activity to remove the RNA of the previously synthesized Okazaki fragment and uses its 5 to 3’ polymerization to replace the RNA nucleotides with DNA nucleotides
Summary of proteins of replication
Sliding clamp
Tethers DNA polymerase 3 to the DNA template making replication more efficient
Summary of proteins of replication
DNA ligase
Seals Nick left between adjacent fragments after RNA primers replaced with DNA
Summary of replication
Each new daughter strand will remain associated with and coiled with the parental strand it was synthesized from
This is because replication of DNA is semi conservative
So in a prophase condensed chromosome, each chromatid consists of one old parental strand and one new daughter strand
Each sister chromatid is an exact copy of the other
In a prophase condensed chromosome each chromatid consists of one old parental strand
Mistakes are very rare in DNA synthesis (high fidelity)
Each replication bubble has 2 expanding replication forks
But these 2 strands are on opposite sides at the 2 forks
Each replication bubble has 2 expanding replication forks, But these 2 strands are on opposite sides at the 2 forks
The chromosomes of eukaryotes are much larger in size and more complicated in structure than prokaryote chromosomes
Eukaryotes chromosomes have many origins of replication to speed up replication (sometimes hundreds of them. Humans estimates have 40-80k)
Expanding replication forks meet along each eukaryotic chromosome to produce a fully replicated chromosome
Bacterial chromosome
Smaller. Simpler. Requires only a single origin of replication called the ori
