DNA Chromo Replication 2c'

Summary

This lecture delves into the intricate structure and replication of DNA, the genetic material. It explains the chromatin structure, highlighting the role of histone proteins in packaging DNA into a compact form. The lecture then transitions into DNA replication, discussing the semiconservative model supported by the Meselson-Stahl experiment. It further explores the process in prokaryotic cells, detailing the initiation at a specific origin, the role of helicases, topoisomerases, and primase in the replication machinery, and the bidirectional nature of the process.

Takeaways

• 🧬 Chromatin structure is composed of DNA and proteins, including histones that form a 'beads on a string' arrangement.
• 🌀 The 30 nm fiber is formed by the coiling of the DNA around histones, which can further organize into loops anchored to the nuclear matrix.
• 🔬 Electron micrographs illustrate the transition from the 30 nm fiber to loop domains, highlighting the role of scaffold proteins.
• 🧪 The Meselson-Stahl experiment provided evidence for semiconservative DNA replication, where each new DNA molecule consists of one old and one new strand.
• 🧵 DNA replication is not random; it initiates at specific origins of replication and proceeds bidirectionally.
• 🔄 Helicases play a crucial role in replication by unwinding the DNA double helix, creating template strands for new strand synthesis.
• 🌀 Topoisomerases relieve the strain caused by the supercoiling of DNA that occurs during replication.
• 🔬 Single-stranded binding proteins prevent reannealing of separated DNA strands and protect them from nucleases.
• 🧬 Primase is essential for replication as it synthesizes RNA primers to which DNA polymerases can add nucleotides, initiating new strand growth.
• 📚 The process of DNA replication is more complex in eukaryotic cells due to larger genomes and involves multiple origins of replication.

Q & A

What is the nucleosome structure described in the lecture?

The nucleosome structure consists of an octomer of four histones forming a 'beads on a string' configuration, where DNA wraps around the histones 1 and 3/4 turns.

What are the two models that explain the further compaction of the 30 nm fiber into a more condensed chromatin structure?

The two models are the solenoid model and the zigzag model, which help to form loops of DNA that are anchored to proteins called a scaffold or nuclear matrix.

What are scaffold attachment regions (SARS) and how do they relate to chromatin structure?

Scaffold attachment regions (SARS), also known as matrix attachment regions (MARs), are the sites where the DNA loops are anchored to the scaffold proteins, contributing to the higher-order folding of chromatin.

How does the removal of histone proteins affect the appearance of DNA as seen under an electron microscope?

Upon removal of histone proteins with high salt, the DNA appears as loose strands that are no longer organized into the structured chromatin fibers, indicating the role of histones in maintaining chromatin structure.

What is the significance of the Meselson-Stahl experiment in understanding DNA replication?

The Meselson-Stahl experiment provided evidence supporting the semiconservative model of DNA replication, where each new DNA molecule consists of one original and one newly synthesized strand.

What are the differences between the three models of DNA replication: semiconservative, conservative, and dispersive?

Semiconservative replication results in each new DNA molecule having one old and one new strand. Conservative replication would result in two old strands staying together and two new strands forming together. Dispersive replication would mix old and new segments in both new DNA molecules.

How does the use of nitrogen isotopes in the Meselson-Stahl experiment help to differentiate between replication models?

The use of nitrogen-15 (heavy) and nitrogen-14 (light) isotopes allowed for the visualization of DNA strands based on their density after centrifugation, which helped to demonstrate that DNA replication is semiconservative.

What is the role of DNA helicase in the process of DNA replication?

DNA helicase unwinds the DNA double helix into single strands by breaking hydrogen bonds between the bases, creating a replication bubble with two replication forks.

Why is topoisomerase necessary during DNA replication?

Topoisomerase is necessary to relieve the strain caused by the supercoiling that occurs as the DNA helicase unwinds the DNA strands, preventing the replication fork from becoming too tight to continue replication.

What is the function of single-stranded binding proteins in DNA replication?

Single-stranded binding proteins bind to the separated DNA strands during replication to prevent their reannealing and degradation, thus maintaining the integrity of the single-stranded DNA template.

Why can't DNA polymerase initiate DNA synthesis on its own, and what is the solution to this limitation?

DNA polymerase cannot initiate DNA synthesis de novo because it requires a pre-existing strand to add nucleotides to. Primase solves this by synthesizing a short RNA primer that provides a starting point for DNA polymerase to begin DNA synthesis.

Outlines

00:00

🧬 DNA Structure and Chromatin Organization

This segment of the lecture delves into the intricate structure of DNA and its packaging into chromatin. The discussion begins with an exploration of chromatin's protein components, specifically the nucleosome, which is an octomer of histones around which DNA wraps in a 'beads on a string' configuration. The lecturer describes the further coiling of this structure into a 30 nm fiber, either through a solenoid or zigzag model, and the formation of loops that are anchored to the nuclear matrix. These loops, which can be up to 85 kilobases in length, are attached by scaffold attachment regions (SARS). The lecture also touches on the visualization of these structures through electron micrographs and the effects of histone removal on DNA structure. Additionally, the use of DNA enzymes to break down and analyze DNA within chromatin is discussed, illustrating the different sized pieces of DNA resulting from various concentrations of the enzyme.

05:00

🔬 DNA Replication: Semiconservative Model and Meselson-Stahl Experiment

The second part of the lecture shifts focus to the process of DNA replication. It emphasizes the importance of accurate replication for genetic material and introduces the concept of semiconservative replication, where each new DNA molecule consists of one original and one newly synthesized strand. The lecture recounts the Meselson-Stahl experiment, which provided evidence for this model. This experiment utilized bacterial cells, nitrogen isotopes (N14 and N15), and equilibrium density gradient centrifugation to track DNA replication over generations. The results, which showed a transition from a 'heavy' DNA band to a 'light' and 'intermediate' band, supported the semiconservative model over conservative or dispersive models. The lecturer also explains the theoretical basis for different replication models and the experimental evidence that led to the acceptance of the semiconservative model as the prevailing mechanism for DNA replication.

10:02

🌟 DNA Replication Process and Initiation

In this section, the lecture provides an in-depth look at the initiation of DNA replication, highlighting the role of specific proteins and the unique starting point known as the origin of replication. It contrasts the replication processes in prokaryotic and eukaryotic cells, noting the differences in speed and the number of origins of replication. The lecture describes the replication apparatus, including the initiator protein (DNA A), helicase (DNA B), and the formation of a replication bubble. It also touches on the role of topoisomerases in managing the strain caused by the unwinding of DNA and the formation of supercoils. The importance of single-stranded binding proteins in preventing reannealing and degradation of the separated DNA strands is also discussed. Lastly, the lecture introduces the concept of primer synthesis by primase, an enzyme that lays down a short RNA primer necessary for DNA polymerases to begin DNA strand elongation.

15:05

🧪 DNA Polymerase and Primer Synthesis in Replication

This segment continues the discussion on DNA replication, focusing on the role of DNA polymerases and the process of primer synthesis. It explains that DNA polymerases cannot initiate new strands but can only add nucleotides to an existing strand, necessitating the use of RNA primers synthesized by primase. The lecture describes the formation of a primosome, a complex of helicase and primase, which is essential for the synthesis of the RNA primer at the replication fork. The process of how DNA polymerases add nucleotides to the 3' end of the primer in a 5' to 3' direction is detailed, illustrating the mechanism of DNA strand elongation. The lecture also includes visual aids to depict the formation of RNA primers and the subsequent DNA synthesis, providing a clear understanding of the molecular events during the initiation phase of DNA replication.

Keywords

💡Chromatin

Chromatin is the complex of DNA and protein found in the cell nucleus. It plays a crucial role in the organization and packaging of DNA within the cell. In the video, chromatin structure is discussed in the context of DNA packaging, where DNA is wrapped around histone proteins to form a compact and organized structure. This is essential for the efficient use of DNA within the cell, as it allows for the regulation of gene expression and the condensation of DNA during cell division.

💡Histones

Histones are a class of proteins that play a central role in the structure of chromatin. They act as spools around which DNA winds, helping to package the DNA into a compact form. In the script, histones are mentioned as part of the nucleosome, which is the basic unit of chromatin. The video describes how histones form an octomer, creating a 'beads on a string' structure that contributes to the higher-order folding of DNA.

💡Nucleosome

A nucleosome is the fundamental unit of chromatin, consisting of a segment of DNA wound around a core of eight histone proteins. The video script describes nucleosomes as the building blocks of chromatin, where DNA wraps around a histone octomer to form a 10-nanometer fiber. This structure is essential for the compaction of DNA within the cell nucleus and is a key concept in understanding how DNA is organized and replicated.

💡Scaffold Proteins

Scaffold proteins are part of the nuclear matrix and serve as anchor points for the chromatin loops. They help in organizing and stabilizing the higher-order structure of chromatin. In the video, scaffold proteins are mentioned as part of the process where DNA loops are anchored to form a more complex structure, which is crucial for the organization of the genome within the nucleus.

💡Semiconservative Replication

Semiconservative replication is a term used to describe the process by which DNA molecules are copied. Each new DNA molecule consists of one original strand and one newly synthesized strand, hence 'semi-conservative'. The video explains this concept by referring to the Meselson-Stahl experiment, which provided evidence that DNA replication is indeed semiconservative, with each daughter DNA molecule containing one old and one new strand.

💡Meselson-Stahl Experiment

The Meselson-Stahl experiment is a landmark study in molecular biology that demonstrated the semiconservative nature of DNA replication. In the video, this experiment is described as a key piece of evidence supporting the semiconservative model, where DNA molecules are replicated such that each daughter molecule contains one old and one new strand. The experiment involved using isotopes of nitrogen to label DNA and observing the resulting patterns after replication.

💡DNA Polymerase

DNA polymerase is an enzyme responsible for synthesizing new DNA strands by adding nucleotides to the growing chain. It plays a critical role in DNA replication. The video script mentions DNA polymerase in the context of the replication process, explaining that it can only add nucleotides to an existing strand, necessitating the use of an RNA primer for initiating new DNA strand synthesis.

💡Primase

Primase is an enzyme that synthesizes a short RNA primer required for the initiation of DNA replication. It works in conjunction with helicase to start the replication process. The video describes primase as being activated by helicase and forming a complex known as a primosome, which is essential for the synthesis of the RNA primer that DNA polymerase will extend.

💡Replication Fork

A replication fork is a Y-shaped region where DNA unwinding and replication occur simultaneously. It is a critical site in the replication process. The video script describes the formation of replication forks as a result of helicase activity, which unwinds the DNA helix, creating single-stranded templates for DNA synthesis to occur in both directions, a process known as bidirectional replication.

💡Topoisomerase

Topoisomerase is an enzyme that relieves the tension in the DNA helix caused by the unwinding action of helicase during DNA replication. It does this by cutting and rejoining the phosphodiester bonds in the DNA backbone. The video explains the role of topoisomerase in preventing the formation of supercoils that could hinder the progression of the replication fork.

💡Single-Stranded Binding Proteins

Single-stranded binding proteins are proteins that bind to single-stranded DNA, preventing it from reannealing or being degraded by nucleases. They play a crucial role in maintaining the stability of the replication fork. The video mentions these proteins as part of the replication machinery, ensuring that the separated strands of DNA remain single-stranded and available for replication.

Highlights

Chromatin structure involves proteins and DNA forming a bead-on-a-string appearance.

The nucleosome octomer consists of four histones creating a 1 and 3/4 turn around the DNA.

The solenoid or zigzag model of chromatin forms a 30 nm fiber.

DNA loops of about 85 kilobases are anchored to proteins known as the scaffold or nuclear matrix.

Loop domains can further fold to create a 700 nm width chromatid.

Histones help maintain the width of chromatin structure.

DNA can be broken down into different sized pieces using DNase enzymes.

Low concentration DNase results in smaller DNA pieces, while high concentration leaves larger pieces.

DNA replication is semiconservative, with one original strand and one new strand in each daughter molecule.

Meselson and Stahl's experiment provided evidence for semiconservative DNA replication using nitrogen isotopes.

Equilibrium density gradient centrifugation was used to analyze DNA replication models.

The experiment showed that DNA replication is not conservative, as no all-heavy band appeared after one generation.

After two generations, the presence of both intermediate and light bands confirmed semiconservative replication.

DNA replication starts at a specific point on the chromosome known as the origin of replication.

Bacterial chromosomes typically have a single origin of replication, while eukaryotic cells have multiple.

DNA replication is bidirectional, proceeding in both directions from the origin.

Initiation of replication involves DNA helicase unwinding the DNA at the origin, forming a replication bubble.

Topoisomerases relieve the strain caused by the supercoiling of DNA during replication.

Single-stranded binding proteins prevent reannealing and degradation of separated DNA strands.

Primase synthesizes an RNA primer required for DNA polymerase to start DNA synthesis.

DNA polymerases can only add nucleotides to the 3' end of an existing strand, not de novo.

Transcripts

all right this is the third part in the

lecture series on DNA genetic material

structure and

replication all right so we going over

chromatin structure talking about the

proteins involved nucleome is at octomer

of these four histones beads on a string

forming 1 and 3/4 turns around seeing

that here that one and 3/4 turns further

uh joining together and either this um

solenoid model or zigzag model creates

what is a 30 NM um fiber here's a

electron micrograph showing that so this

30 nanm chromogen fiber then can form

these Loops like I said about 85

kilobases of DNA and these are anchored

to um proteins called a scaffold or

nuclear Matrix the loops are attached to

the scaffold by scaffold um attachment

regions or SARS scaffold attachment

regions or Matrix attachment region or s

m RS these Loop domains can f can

further fold to create a 700 nanm width

chromatid um so if we look down here the

chromatid which is half of this so the

width from here to here is about 700 um

nanometers and so here they've sort of

digested away the histone protein so all

the DNA is just loose see DNA strand

here and it just tons of DNA just looped

here so all these loops and then they're

attached to the scaffold proteins so

this red stuff in here is scaffold

proteins and again so high salt removed

the histone protein so that stuff went

away but it was still connected to these

scaffold

proteins and so the uh hison then help

keep it at about this width and you can

see if the histones are gone they sort

of unravels and goes everywhere um so

here we can say um a loop um bound to

nuclear

Matrix filament so here you got this

Loop this 30 nanom nanometer Loop here

and you got these attachment regions

that attach to these Matrix attachment

regions um and here you can see a

closeup a nuclear Matrix just this and

again it's called a matrix because

they're just joined in all these um

different parts and here's the nuclear

Matrix and here joined in all these

different

areas and here's a um way to sort of

break DNA down into different sized

pieces as it's contained in chromatin so

you can use DNA one which is just an

enzyme that chops up DNA you can use a

low concentration DNA Swan up to a high

concentration DNA Swan and then uh run

those out on gel and the idea is that so

before digestion you got a lot of DNA

long pieces join beats on the string

after DNA is use a Cuts between all

these nucleosomes so you get these

different sized pieces the bigger pie

pieces stay close to where they started

smaller pieces go further down so if you

then look at the actual results of this

if you got a low amount of DN some of

the DNA gets cut into really small

pieces but there's big chunks of DNA

because there's not a lot of DNA so some

of that DNA isn't cut so you get big

long pieces medium amount you get more

smaller pieces and you're missing larger

pieces and a high amount of DNA Cuts

almost all the DNA into then um smaller

pieces that can be seen here so just a

summary of you know what we're looking

at here so DNA without any um protein

DNA in um those histone octomer

nucleosomes wrapped around um those

rolling up so that's 10 10 net wide or

11 um rolling up to 30 NM fiber solonoid

structure or a zigzag model so you get

this 30 big Loops then contained or

joined to these matrices or M

in the um nucleus and then those even

further Loop up so that here we go the

uh width here is 700 nimet so one of

these right here that's 700 net so both

of them together

is400 um

together all right so that's uh keriton

structure now we're going to move on and

talk to DNA replication or how DNA makes

an identical copy of itself so the

double helix model of DNA that Watt and

Crick proposed made it immediately

evident to them a mechanism for DNA

replication in fact they included a

quote in their paper saying as much so

this comes directly um from their paper

their study um talking about the

structured DNA and they say quote it has

not escaped our notice that the specific

pairing we have postulated immediately

suggests a possible copying mechanism

for the genetic material unquote so they

sort of slightly state that you know

this model they came up with really

explains how DNA could replicate itself

quite easily so recall that the ability

to replicate information Faithfully is

one of the requirements for the genetic

material because the two strands are

complimentary to one another one can

imagine the strands separating and each

New Strand is red or replicated from the

information contained on the original

strand this type of replication is

called

semiconservative replication because one

conserved strand stays and and one New

Strand is made there are however other

possible models of DNA replication

conservative and

dispersive the mesel install experiment

provided evidence to support the

semiconservative um semiconservative

replication for DNA so in 1958 mes estal

studied DNA replication using bacterial

cells isotopes of nitrogen nitrogen 14

and nitrogen 15 so these are naturally

occurring Isotopes they are not

radioisotopes they just differ in Mass

so natural isotope 14 and a heavier

isotope N5 and equilibrium density

gradient centrifugation okay so again

Isotopes same atomic number but

different atomic mass making N15

slightly having slightly more mass than

n14 so equilibrium density graded

equilibrium density gradient

centrifugation is centrifugation of

heavy Salt Solutions in this case they

use cium chloride which has a molecul

133 which you don't need to remember um

the heavy

cium during centrifugal forces um tends

to sediment towards the bottom of the

tube however diffusion also takes place

during centrifugation so you have is two

forces that work and after long periods

of centrifugation at high speeds a

linear concentration gradient of cium

chloride is formed in the centrifugation

tube such that you have a higher

concentration at the bottom and then

diffusion creates a gradient As you move

toward the top of the

tube macro molecules then so protein DNA

RNA in the tube will come to rest or

will band in the region of the tube

where their density matches the density

of the cium chloride okay and again the

density of cium chloride goes from high

at the bottom of the tube to lower lower

lower lower lower As you move toward the

top of the tube so chloride is used

for this sucrose can also be used or

sucrose gradients can also be used to um

in this type of um centrifugation so

what was the

experiment um let see oh back up here so

a couple of notes before we get to the

experiment um so this is

semiconservative replication displayed

here so again you start with one strand

of DNA you end up with two strands of

DNA it's semiconservative because this

strand was conserved and this New Strand

was made well this strand on this side

was conserved and this strand was made

so you can see if you could separate

these two strands then make a new strand

that would be a semiconservative model

however you could also Imagine different

models so semiconservative in the middle

which just talked about you could have a

conservative model of replication where

two strands of DNA stay together and two

brand new strands are made together or

you could have a dispersive model where

you have two strands of DNA where parts

of both sides of the strands are new and

old so you can see mixed here so you get

a mix in both of the strands rather than

one purple one blue you get a mix of

purple and blue on both sides here so

those are the models of replication so

what did meselson install do then to

determine what was going on so cells

were grown in a medium containing heavy

nitrogen Okay so in this case suspension

of bacterial cells labeled with

N15 okay so the bacterial cells were

grow grown in N5 for several Generations

so that all the nitrogen in the DNA

contained heavy nitrogen cells were

washed to remove N15 and transfer to

Media containing

n14 okay so it's added to

n14 cells were allowed to grow in the

n14 for various time periods either one

or two cell

divisions and then their DNA was

extracted and analyzed by cium chloride

equilibrium density centrifugation so

again here all Heavy added to n14

allowed to divide either once or allowed

to divide twice and they went further

than that but we don't need to to

explain what's happening and then they

would lice okay or break apart the cells

they would get the DNA out and then they

would run it on this so they put on the

CM chloride gradient here get this and

they would spin it and the DNA then

would run you can see barely see there's

bands here so you either get heavier or

lighter or lighter size bands

as the DNA went along so here's our

actual um data from their paper you can

see 1958 M

install

and generations after adding in14 so

actually move some right to left here so

here's one generation so half heavy

heavy so what they do is they start with

all Heavy because they're all in n14 and

you get a band here after one generation

here all the DNA is half heavy or a mix

of n14 and N15 if you then jump to this

is about two generations what you have

is half heavy or a mix of n14 and15 and

light or about equal amounts of mix and

all light and you can see here as the

keep going Generations further and

further out you get more and more of

this light but you never lose this band

here of the half heavy but you do lose

the heavy right you never get another

Heavy band which immediately after one

generation this says that that

conservative model was not um the model

by which T replicates because you would

expect a band oops out to one here you'd

expect a band but it's not there

um the half Heavy after one generation

explains the um cons uh semiconservative

model but it also explains possibly the

dispersive model but if you go out two

generations here dispersive model you

should only get one band right about the

middle of here but instead you get two

bands one with the half heavy and one

with the light and so by going out two

generations that tells you that is the

semiconservative model of replication um

is how DNA replicates

itself all right so that explains what

happens but um if it's not completely

clear you probably want to take this

model here label both these purple

strands and5 and H blue strand that

forms and14 and make sure that um you

can sort out what would happen if you

centrifuge each of these bands of DNA

where would they band so what ends up

happening is this is The Heavy band this

is the intermediate band there's an

intermediate band there's an

intermediate band these then are the

light bands so that's why after one

generation you see nothing but

intermediate at two generations you see

an equal amount of intermediate

intermediate and light because there's

two light complete lights and two

intermediates on the end all right so we

know that DNA replication is

semi-conservative now we'll talk about

how DNA actually replicates itself and

we'll start with just a few General

statements um so while a lot is known a

lot is still unknown um much more um is

known about procaryotic DNA replication

than eukariotic DNA replication so the

discussion of replication we'll center

around the results of procaryotes uh

since they are known earlier and known

better and then we'll discuss um some

issues about replication UK carots

there's lots of things that are similar

and there's just details um that are

different so replication of DNA does not

start at a random Point okay the origin

of replication is a unique point along

the chromosome where replication begins

this is a short sequence recognized by

the replication apparatus as the point

to begin rarely does replication begin

at the end of a chromosome and of course

bacterial chromosomes are circle and

have or circular and have no free ends

bacterial chromosomes usually around up

to a million base Bears have a single

origin of

replication the rate of replication in

these organisms is about 2,000 nuclear

Tides per second um an ecoli cell can

replicate its DNA between 20 to 40

minutes eukariotic replication is much

slower between 2,000 to 10,000 base

pairs per minute and remember 2,000

nucleotides per second for bacterial

cells 2,000 to 10,000 base Bears per

minute for eukariotic cells due to the

slow speed of replication and larger

number of base pairs in ukar ukar have

several origins of replication each

around 60,000 base pairs apart a yeast

cell can replicate its DNA in about 1.4

hours and animal cells and culture may

take up to 24 hours to replicate their

DNA a Replicon is the origin of

replication Replicon is the origin

replication and the DNA that is

synthesized from the origin DNA

replication is nearly always bio sorry

bidirectional um meaning that

replication proceeds in both directions

along the nucleotide strands so what

that would look like here so if there's

an origin replication here DNA

replication goes to the left and to the

right okay so bir directional moving at

moving in both directions and so here's

some evidence of that started here moves

this way so it's making conservative

semiconservative um there and also in

these little Loops all right and so

here's an electrom micrograph sort of

showing that same

thing um here's chromosome after one

round of replication here chromosome

undergoing its second round

replication there

oops um so it's moving in both

directions going to the left and to the

right all right so details of

replication from E coli or um bacterial

cells initiation replication begins at

the origin replication an initiator

protein called DNA

a is required to recognize and bind the

sequence of nucleotides at the origin of

replication this recruits DNA

b a protein containing two helicases so

a helicase is an enzyme that unwinds DNA

remember DNA is a helix a double helix

so a helicase unwinds the DNA in

um at the origin to denat the DNA double

helix into single strands and form

what's called a replication bubble

untwisted single strands from which new

strands are made are called template

strands okay so when the he case comes

in it separates the double strand into

individual strands those individual

strands then are known as template

strands all right so here's or C or

origin replication E coli and um you can

see it has this specific region here and

region here this then the sequences are

what are recognized by that DNA a uh

protein and you can see here this is

called the DNA a box often times um

scientists you know geneticist call

sequences of DNA that are bound by

protein boxes okay so this is a box or

it's a sequence of DNA and it's a DNA a

box because DNA a is the protein that

binds to that particular sequence so the

origin replication you don't need to

know any of these sequences it's just

the spot where DNA a will bind that

origin

replication all right so so you have

these template strands the rest of the

replication apparatus which contains

several parts in one multi-subunit

apparatus is called the

reome um now we'll join that replication

bubble so initiator protein recognizes

recruits helic cas which um opens up

single strands that creates that

replication bubble or two replication

forks all the other components then that

join all together then known as the

repone all right so then here's just

another figure those DNA boxes here so

DNA protein can bind there it's going to

recruit them the DNA helicases or DNA B

that can come in and those are going to

open so this purple double stranded open

into single strand Brands creating this

replication bubble or two replication

forks so to the right and then to the

left a by directional replication that's

going to take place um this is sort of

advanced we haven't gotten to these

details but same sort of thing so this

now is a replication fork it's Bas

basically if you if you cut this in half

down where this arrow is and you just

looked at one side of it you'd have a

replication fork rather than a bubble so

two replication forks make up a bubble

so here's replication Fork the fork is

moving to the left the purple on the top

new blue being made purple on the bottom

new blue also being made so

semiconservative replication we'll get

to these details here in a

second okay so let's talk about the

details of the initiation or the start

of DNA replication so as that DNA

helicase unwinds the DNA at the growing

Fork it does so by breaking apart the

hydrogen bonds between the bases this

requires ATP so it requires some energy

to do the job this unwinding the DNA by

DNA hel case also causes extra twisting

in other regions and coils called super

coils form to release the strain these

super coils then that form are relieved

or removed by topoisomerases so

topoisomerases are enzymes that control

twisting of DNA and they are needed to

break phosphodiester bonds and twist the

DNA Helix back back to what's called its

relaxed State and join them DNA gyas is

one example which can induce super

coiling of DNA so take a look at

this here so as these strands open up so

D hel cases separating these two strands

what ends up happening is there's extra

twisting that happens here and you get

these super coils well if this was

allowed to continue and this opened up

further and further this super coiling

region would be so tight that the

replication fork would not be able to

open up any further so you need these

top isomerases to cut and unwind these

regions and so if you look then don't

you don't have to look at this one here

I don't think it's going to show up but

if you look at these YouTube videos here

they do a really good job of showing how

these Topo isomerases work to relieve

this um the super coiling and the

structural pressure that's um being

created through this

process okay so the strands are being uh

pulled apart or opened up by the

helicases the top isomerases are taking

care of the super coiling single

stranded binding proteins um bind single

stranded DNA preventing Its reannealing

So once um those strands are separated

you don't want them to anneal or come

back together you also don't want them

to um be chewed apart by any nucleases

within the cell so single strand binding

protein binds to them prevents them from

binding back together and they also

prevent them from being degraded so the

next thing then that happens is primer

synthesis so it turns out that DNA

polymerases and we haven't got that part

yet they're part of the replisome

polymerases polymerize or make and put

DNA together um DNA polymerases that are

responsible for DNA synthesis cannot

catalyze the addition of

nucleotides denovo or all by themselves

they can only add nucleotides to the

exist to a pre-existing strand so there

has to be a strand of nucleotides there

for DNA Pates to add nucleotides to to

grow they can't put that first

nucleotide in brand new they're unable

to do that so therefore how the cell

deals with this is there's an enzyme

called

primase right here that synthesizes a

short RNA primer which is complementary

to the DNA DNA template strand DNA plas

can then add nucleotides to the existing

primer so primase is activated by

helicase so again helicase is already

opening the strands up and they form a

complex with the DNA called a primosome

all right so the primosome then is the

helicase plus primase helps form the RNA

primer so let's take a look at what that

looks like all right so here the DNA is

replication fork is being separated and

as I stated DNA plase can't put in this

first nucleotide all on its own so what

has ends up happening is the primer gets

made by primase and it's complimentary

to the DNA it's there now DNA plyar can

come in and put this blue nucleotide and

it can add a nucleotide to the end of an

existing DNA strand all right and what

we're going to talk about in a few

minutes but since we have the SL up is

DNA plase adds nucleotides to the thre

Prime end of an existing strand so this

strand is five Prime to three prime it's

going to add nucleotides to the three

prime end it cannot DNA polymerases

cannot add nucleotides to the five Prime

end it's just how it works and it can't

do that so

um polymerises DNA pises always create

or grow a DNA strand in a direction five

Prime to three prime it can't do so in

the other direction all right so here's

a slide here here in red is a primer

actually you can see it here better an

RNA primer and this blue is new DNA that

was made here the red is primary here's

a blue and here's the enzymes making it

making it this point here's a new RNA

primer that was put in and we'll start

um DNA plas will be made from that here

again primase making an RNA primer and

then the DNA plases will make the DNA so

here's an RNA primer in red DNA's been

made joins up here's another RNA primer

you see the end of it in red going to be

used to make that so we're going to end

here and then we'll pick up right at

this point to continue talking about how

uh DNA replication works