DNA Replication 2d'

Summary

This lecture delves into DNA replication, focusing on the role of primers and DNA polymerases in the process. It explains that DNA polymerases, including DNA polymerase 1, 2, and 3, synthesize DNA in a 5' to 3' direction, requiring a pre-existing strand. DNA polymerase 3 is highlighted as the primary enzyme for chain elongation, possessing proofreading capabilities to ensure accuracy. The lecture also touches on the removal of RNA primers by DNA polymerase 1 and the joining of Okazaki fragments by DNA ligase. It contrasts prokaryotic and eukaryotic DNA replication, noting the involvement of more enzymes and the presence of telomeres in the latter, which are maintained by the enzyme telomerase to prevent shortening during cell division.

Takeaways

• 😀 DNA replication requires a primer, which is an RNA molecule that provides a starting point for DNA polymerase to add nucleotides.
• 🔬 Bacterial cells have five DNA polymerases, with polymerases 1, 2, and 3 being the most studied and essential for DNA replication.
• 🧬 All three main bacterial DNA polymerases synthesize DNA in a 5' to 3' direction and require a free 3' hydroxyl group to add the next nucleotide.
• 🔄 DNA polymerase 2 has a less clear role but is involved in DNA repair, while DNA polymerase 3 is the primary enzyme for chain elongation during replication.
• 🔍 DNA polymerase 1 is responsible for degrading the RNA primer and filling in the gap with DNA, as well as participating in general DNA repair.
• 🔗 DNA polymerases have proofreading capabilities, which improve the fidelity of DNA replication by correcting incorrect nucleotides.
• 🌐 DNA replication in vitro can be achieved with the right components: deoxynucleoside triphosphates, magnesium, a template, and a primer sequence, along with DNA polymerase 3.
• 🔁 The process involves the removal of RNA primers by DNA polymerase 1 and the joining of Okazaki fragments by DNA ligase to form continuous DNA strands.
• 📚 Eukaryotic DNA replication is similar to prokaryotic but involves more factors, including multiple origins of replication and the need to deal with nucleosomes and chromatin structure.
• 🧵 Telomeres at the ends of chromosomes pose a problem for DNA replication, but the enzyme telomerase helps maintain chromosome length by adding telomeric repeats.

Q & A

What is the role of a primer in DNA replication?

A primer is necessary for DNA polymerase to add nucleotides to the growing DNA strand. It provides a starting point with a free 3' hydroxyl group for the DNA polymerase to attach the next nucleotide.

How many DNA polymerases are involved in DNA replication in E. coli?

There are five DNA polymerases in E. coli, but the best studied ones for DNA replication are polymerase I, II, and III.

In what direction do DNA polymerases synthesize DNA?

DNA polymerases synthesize DNA in the 5' to 3' direction, meaning they can only add nucleotides to the 3' end of the growing strand.

What is the function of DNA polymerase III in E. coli?

DNA polymerase III is the primary enzyme involved in DNA replication and chain elongation in E. coli. It is responsible for the majority of the DNA synthesis during replication.

What is the role of DNA polymerase I in DNA replication?

DNA polymerase I is responsible for degrading the RNA primer and filling in the gap with DNA, thus replacing the RNA primer with DNA.

What is the purpose of the 3' to 5' exonuclease activity in DNA polymerases?

The 3' to 5' exonuclease activity allows DNA polymerases to correct errors by removing incorrect nucleotides that were added in the 3' to 5' direction, thus acting as a proofreading function.

How does DNA polymerase III ensure high fidelity during replication?

DNA polymerase III ensures high fidelity through its proofreading function, which involves the 3' to 5' exonuclease activity to remove incorrect nucleotides and its active site that checks the diameter of the DNA for correct base pairing.

What are Okazaki fragments and how are they formed?

Okazaki fragments are short segments of DNA that are synthesized on the lagging strand during DNA replication. They are formed due to the discontinuous nature of lagging strand synthesis, which requires the use of multiple RNA primers that are later replaced by DNA.

What is the role of DNA ligase in DNA replication?

DNA ligase seals the nicks between Okazaki fragments by forming a phosphodiester bond between the 3' hydroxyl group on one fragment and the 5' phosphate group on the adjacent fragment, thus joining the fragments together.

How is DNA replication initiated in eukaryotic cells?

In eukaryotic cells, DNA replication is initiated at multiple origins of replication by the action of proteins such as the origin recognition complex (ORC), Cdc6, and Cdt1, which function as helicases to unwind the DNA.

What is the function of telomerase and why is it important for maintaining chromosome ends?

Telomerase is an enzyme that adds telomeric repeats to the ends of chromosomes, preventing them from getting shorter with each cell division, which would otherwise occur due to the inability of DNA polymerase to fully replicate the ends of linear DNA molecules.

Outlines

00:00

🧬 DNA Polymerases and Replication Process

This segment delves into the intricacies of DNA replication, focusing on the role of primers and DNA polymerases. It explains that DNA polymerases, such as those found in E. coli (Pol I, II, and III), synthesize DNA in a 5' to 3' direction, requiring a pre-existing strand and an RNA primer with a free 3' hydroxyl group. Pol III is highlighted as the primary enzyme for chain elongation, while Pol I removes the RNA primer and fills the gap with DNA. The lecture also touches on the exonuclease activity of these polymerases, which allows them to correct errors by moving in a 3' to 5' direction. The discussion wraps up with an overview of how DNA polymerase III, as part of the holoenzyme, manages the replication of both the leading and lagging strands with high fidelity, thanks to its proofreading function.

05:01

🔬 In Vitro DNA Synthesis and Replication Mechanisms

The second paragraph shifts the focus to how DNA can be synthesized in vitro, requiring only four deoxynucleoside triphosphates, magnesium, a template, and a primer. It illustrates the process of DNA strand elongation, where nucleotides are added to the growing strand in a 5' to 3' direction, and the energy for bond formation comes from the cleavage of pyrophosphate from the nucleoside triphosphates. The paragraph also describes the formation of replication bubbles and forks, explaining the continuous synthesis on the leading strand versus the discontinuous synthesis on the lagging strand, resulting in Okazaki fragments. These fragments, along with the removal of RNA primers, are set up to be joined together, hinting at the role of DNA ligase in the subsequent steps of the replication process.

10:05

🧩 Okazaki Fragments and DNA Ligase Function

This section hones in on the removal of RNA primers and the joining of Okazaki fragments, critical steps in the DNA replication process. DNA polymerase I is depicted as the enzyme responsible for degrading the RNA primer and replacing it with DNA, leveraging the adjacent pre-existing strand. However, the Okazaki fragments produced on the lagging strand are not initially covalently bonded to each other. The role of DNA ligase is then introduced, which seals the fragments together by forming phosphodiester bonds between the 3' hydroxyl group of one fragment and the 5' phosphate group of the adjacent fragment. The paragraph visually describes this process using replication forks and the action of various enzymes, culminating in the seamless joining of DNA fragments.

15:08

🌐 Eukaryotic DNA Replication: A Closer Look

The lecture now contrasts prokaryotic DNA replication with that of eukaryotes, noting similarities and key differences. Eukaryotic replication is more complex, involving more proteins and enzymes, and is initiated at multiple origins by an origin recognition complex (ORC) and helicases like Cdc6 and Cdt1. The process is slower, and the Okazaki fragments are smaller, approximately 135 nucleotides long. Additionally, eukaryotes must manage the chromatin structure, involving histone proteins, which are disassembled and reassembled during replication. The paragraph also touches on the role of chromatin assembly factor 1 (CAF-1) in histone targeting and the significance of DNA replication occurring during the S phase of the cell cycle.

20:08

🧵 Telomere Maintenance and the Role of Telomerase

The final paragraph addresses the unique challenge of replicating the ends of chromosomes, known as telomeres. It explains the problem of chromosome shortening due to the inability of DNA polymerase to replicate the very ends of linear DNA molecules, which could lead to cellular aging. The paragraph introduces telomerase, an enzyme that counters this shortening by adding telomeric repeats to the chromosome ends. Telomerase consists of an RNA component that base pairs with the telomeric DNA and an enzymatic component that extends the DNA strand using the RNA as a template. This process is crucial in cells that undergo many divisions, such as germ cells and stem cells, and is linked to cellular immortality observed in tumor cells. The lecture concludes with an overview of the DNA replication process, highlighting the various enzymes and their functions in both prokaryotic and eukaryotic cells.

Keywords

💡DNA polymerase

DNA polymerases are enzymes that play a crucial role in DNA replication by synthesizing new DNA strands. In the script, DNA polymerases are highlighted as essential for adding nucleotides to the growing DNA chain in a 5' to 3' direction. They require a pre-existing strand with a free 3' hydroxyl group to add the next nucleotide. The script mentions three main DNA polymerases in bacteria: DNA polymerase I, II, and III, each with distinct functions. DNA polymerase III, for instance, is the primary enzyme for chain elongation during DNA replication.

💡Primer

A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. In the context of the video, primers are necessary for DNA polymerases to begin the addition of nucleotides to the DNA strand. The script explains that DNA polymerases require an RNA primer to initiate replication, and these primers are later replaced by DNA by the action of DNA polymerase I.

💡Replication fork

The replication fork is a Y-shaped structure formed when the two strands of a DNA molecule are separated in preparation for replication. The script describes how helicases open up the DNA to form a replication bubble, which is then split into two replication forks. These forks are the sites where DNA synthesis occurs, with one strand being synthesized continuously (leading strand) and the other in fragments (lagging strand).

💡Leading strand

The leading strand is the DNA strand that is synthesized continuously in the 5' to 3' direction as the replication fork moves. The script illustrates this concept by showing how the leading strand grows without interruption, as opposed to the lagging strand, which is synthesized in discontinuous fragments known as Okazaki fragments.

💡Lagging strand

The lagging strand is the DNA strand that is synthesized in short fragments, called Okazaki fragments, because it is moving in the 3' to 5' direction, opposite to the movement of the replication fork. The script explains that the lagging strand requires multiple RNA primers as the replication machinery moves along, creating these fragments that are later joined together.

💡Okazaki fragments

Okazaki fragments are the short segments of DNA on the lagging strand that are synthesized in the 5' to 3' direction as the replication fork moves. The script mentions that these fragments are named after their discoverer and are joined together by DNA ligase after the RNA primers are removed by DNA polymerase I.

💡DNA ligase

DNA ligase is an enzyme that joins DNA fragments together by forming a phosphodiester bond. In the script, DNA ligase is described as sealing the nicks between Okazaki fragments on the lagging strand, creating a continuous DNA strand. This enzyme is crucial for completing the replication process by ensuring there are no gaps in the newly synthesized DNA.

💡Exonuclease activity

Exonuclease activity refers to the ability of some enzymes to break down DNA or RNA from the end of a nucleic acid strand. The script highlights that DNA polymerases have 3' to 5' exonuclease activity, which allows them to remove incorrect nucleotides that have been added in the 3' to 5' direction, contributing to the proofreading function of DNA polymerase III.

💡Proofreading function

The proofreading function of DNA polymerases is a mechanism by which they can correct errors during DNA replication. The script explains that DNA polymerase III, for example, has a proofreading function that allows it to detect and remove incorrect nucleotides, thus increasing the fidelity of DNA replication.

💡Telomerase

Telomerase is an enzyme that maintains the length of linear chromosomes by adding telomeric DNA to the ends. The script discusses the role of telomerase in adding TTAGGG repeats to the ends of chromosomes, which is crucial for preventing the loss of genetic information during replication. This enzyme is particularly important in cells that undergo many divisions, such as stem cells and germ cells.

Highlights

DNA polymerases are essential for DNA replication, with E. coli cells having five polymerases, primarily DNA polymerases I, II, and III.

All three major DNA polymerases synthesize DNA in a 5' to 3' direction, requiring a pre-existing strand and a free 3' hydroxyl group.

DNA polymerase II's role is unclear but is involved in repair, while DNA polymerase III is the major enzyme for DNA replication and chain elongation.

DNA polymerase I is the most active and is responsible for degrading the RNA primer and filling in the gap with DNA.

DNA polymerases possess 3' to 5' exonuclease activity, allowing them to correct errors by removing nucleotides in a reverse direction.

DNA polymerase III has a proofreading function, which significantly improves its fidelity, making mistakes less than one in 10 billion nucleotides.

The enzyme works by forming a cylinder around the DNA, which aids in proofreading by sensing the diameter of the DNA helix.

DNA replication involves the synthesis of the leading and lagging strands, with the latter being discontinuous and known as Okazaki fragments.

The joining of Okazaki fragments is facilitated by DNA ligase, which forms a phosphodiester bond between the fragments.

In vitro DNA replication is possible with the right components, including deoxynucleoside triphosphates, magnesium, a template, and a primer sequence.

Eukaryotic DNA replication is similar to prokaryotic, but with more factors involved and a slower process.

Eukaryotes have multiple DNA polymerases, with replication occurring during the S phase of the cell cycle.

Nucleosomes must be disassembled and reassembled during eukaryotic DNA replication, involving proteins like chromatin assembly factor one.

Telomerase is a unique enzyme that maintains chromosomal lengths by adding telomeric repeats to chromosome ends.

Telomerase is composed of both RNA and protein, with the RNA component being complementary to the telomeric repeat unit in the DNA.

The process of telomere replication involves the extension of the DNA strand by telomerase, allowing for the maintenance of chromosome length.

Transcripts

okay hello again this is the fourth part

of the lecture series on DNA the genetic

material structure and

replication okay so we left off talking

about primer synthesis how a primer

needs to be present in order for DNA

plase to add nucleotides to that and we

looked at a couple of different slides

this one and this one here pointing that

out so what happens after um the primer

is there well there are different DNA

polymerases that synthesize DNA so eoli

or bacterial cells have five polymerases

for DNA replication we'll talk about the

best studies of those the best studied

of those which are plase 1 2 and three

all three of these DNA plases synthesize

DNA in a five to three prime Direction

okay so they can only add nucleotides

onto that three prime end so they have

to move from five

2 3 Prime um and all three of them

require a pre-existing strand or that

RNA primer has to be there okay and it

has to then have a free three prime

hydroxy group to add that next

nucleotide to um so to some sorry and

the third thing is they have three prime

five Prime

exonuclease Direction but they can

actually back up an excle or take out

nucleotide es sort of in Reverse are in

a three prime to five Prime Direction so

DNA poies 2 um it's precise rolls

unclear but is probably involved in

repair so repairing broken parts of DNA

it can come in and and put the right or

replace nucleotides DNA plase 3 is the

major enzyme involved in DNA replication

and chain elongation there's only about

10 to 20 molecules of DNA plase 3 per

cell and it breaks down quite easily um

this was discovered in 1971 so actually

relatively late and they're named in the

order of their Discovery so one was

discovered first two and then three D

plas 1 was the first plase discovered in

1959 and it is the most active plase

there's about 400 copies of it per cell

and its job is to degrade the RNA primer

and fill in that Gap where the RNA

primer was with DNA and we'll walk

through this so that makes sense um it

was also involved in general U DNA

repair so and again as I pointed out in

addition to this five Prime to 3 Prime

polymerase activity these enzymes have 3

to 5 Prime Exon nucleus activity all

right so DNA plas 3 is the primary chain

elongation enzyme it also has

exonuclease activity as we talked about

in a 3 to five Prime Direction but it

can also

with a new nucleotide um this the

polymerase and the exonuclease

activities are found in the same active

site of the enzyme and it works by

forming a cylinder around the DNA and

you saw this in the figures we'll show

the figures again it sort of surrounds

the entire double stranded DNA or at

that point open single stranded DNA in

order to add

nucleotides um and part of the proof

reading function comes from this

cylinder that forms around the DNA as it

uh that cylinder has a certain diameter

that as DNA passes through it the enzyme

can measure the diameter of DNA and if

an incorrect nucleotide is put in

especially if it's a purine with a

purine that or a perine with a perine

either of those it can sense um the

wrong diameter and it knows it's put the

wrong um nucleotide in uh DNA plase 3 is

part of What's called the larger DNA

polymerase 3 Holo enzyme which is part

of that

reome and it contains two catalytic

cores one for the leading Strand and one

for the lagging strand

and this is really difficult to sort of

picture the leading and lagging strand

being used all at the same time I'll

sort of show it as we'll look at one

strand at a time it's just easier to see

but this enzyme actually has two

catalytic cores it's able to do both

strands at the same time um even though

it's got a proof reading function and it

has high fidelity meaning it barely

makes mistakes it still occasionally

makes mistakes uh but less than one in

10 billion nucleotides is wrong um and

that's with the proof reading function

if it didn't have a proof reading

function it would make an error about

once every million times so proof

reading actually really improves its

Fidelity all right so this one's kind of

funny to uh sort of see but if you look

at this enzyme here this blue is what's

growing and it's growing in a five to

three prime Direction so the five Prime

end is here so this part of DNA

polymerase 3 is adding nucleotides at

this blue end and it will continue from

left to right and this one here it's

sort of Twisted the DNA around so it's

still going left to right but it's still

growing here's the end of it five Prime

to three prime okay so it's still adding

in the same direction eventually it'll

run into this strand DNA that's here so

that blue will run into the red and

we'll talk a little bit later that RNA

primer will need to be removed and

replaced um this figure very similar

shows the DNA sort of looping around so

you're growing from here they don't have

five Prime and three prime well you can

find them up here but growing in it'll

join there this one will grow and join

there this is then a leading strand this

goes around it's growing from five Prime

down in the bottom here to three prime

up in that

direction all right so we'll get back to

that in some of the extra steps but it

turns out that from what we know about

um how um replication works that we can

make DNA in vitro or in a test tube and

this is done a lot in Labs all around

the world and you only need a few things

so the things that you need are all four

deoxynucleoside triphosphates so the

building blocks you need some magnesium

which helps DNA plas work you need a

template or you need something for the

pmer to read in order to put in the

correct nucleotides and you need a

primer sequence just like you need an

RNA primer in our cells you need an

artificial primer sequence to put into

the tube and you need DNA plase 3 or one

it turns out in vure on the test tube

that DNA plase 3 can do the job and is

actually the one that um most often is

used because it was discovered first but

DNA plas one which works in our cells um

can also be used to make DNA in vitro

all right here so here we show the

joining of a new nucleotide onto an N so

here's um the original DNA strand here

in purple and here's in blue the growing

Strand and you can see here right

there's a nucleoside mono phosphate

that's what's in the DNA but the monomer

units use their nucleoside triphosphates

and the reason for this is as this

triphosphate comes in there needs to be

energy in order to produce this bond

between this oxygen and this

mainly oxygen and the phosphate

containing group so what ends up

happening is two of these phosphate

groups are cleaved off right here and

they are called pyrophosphate and the

energy from that cleavage then is used

to create this calent Bond joining the

next nucleotide together so when um I

said in the last slide there we needed

nucleotide triphosphates you need those

two extra phosphates for the energy in

order to catalyze reaction to joining

those nucle tied together and here's

just sort of a schematic with letters

showing the same thing um TG this T is

coming in it's growing from left to

right cleave the two phosphates create

that Bond and the pyrophosphate is then

released all right so um some more

information on this um so as I mentioned

before when those helicases open up the

DNA it forms a replication bubble if you

split that replication bubble right in

half you create a replication fork on

the left and a replication Fork on the

right so two forks together make the

replication bubble um and again that DNA

replicates in both directions okay to

the left and to the right and DNA

polymerase works by adding nucleotides

from the five Prime to three prime

Direction and so replication bubbles one

big bubble the forks then move in One

Direction so the fork on the left will

move to the left the fork on the right

we'll move to the right um for

Simplicity we'll generally just look at

a single rep application Fork which

you'll see in the next few um images so

DNA strands are anti parallel synthesis

only occurs in the 5 to three prime

Direction um when both DNA strands

separate replication still occurs only

in the five to three prime Direction so

one strand with the replication fork

moving in the template strand Direction

3 to 5 Prime so one of those strands is

synthesized without interruption in a 5

to3 Prime Direction This is called the

leading Strand and we saw this in this

image here here so this you see five

Prime you see it's continuous it's

unbroken okay and that is the leading

strand it is made in one long unbroken

strand so here's the the fork so here's

the DNA together here's it separated to

the left and to the right so it's sort

of a two pronged Fork here on the left

side is the leading strand because it's

made continuously on the other side you

get the lagging strand you get a strand

made here see it ends here's another

strand just about joining it this strand

is going to grow and you're going to

join it you a bunch of discontinuous

pieces of DNA that are made that run

into the primers continuously and we'll

talk about how that's fixed but those

then are called the leading or sorry the

lagging strand so if the leading strand

is continuous the lagging strand is dis

continuous so these lagging strands then

are known as okazaki fragments okazaki

named after the person who first

discovered them and there are about

1,000 to 2,000 nucleotides in

procaryotes okay and they're a little

bit shorter than in ukar so how then do

those okazaki fragments get joined

together and first really is how do the

RNA primers get removed and then how do

those um okazaki fragments get put

together we'll talk about this in a

second first we'll go over to a couple

of images here showing leading and

lagging again so again here's a

replication bubble on the bottom here

the leading strand is in blue because

it's continuous so that means on the top

the the leading strand goes in the

opposite direction because of the five

Prime and three prime there's no five

Prime and three Prime on here uh a good

thing for you to do you is you should be

able to label the five and three Prime

on all these strands the legging strand

then on the top goes from left to right

the lagging strands on the other side go

from right to left and those then are

known as okazaki fragments if we look at

it in a fork again five Prime thre Prime

so five Prime here there would be a

primer here it grows continuously as a

leading strand so on the other side then

has to grow five Prime to 3 Prime five

Prime to 3 Prime five Prime to three

prime so it keeps running into they

don't show the RNA primers here but

those RNA primers would be on the five

Prime and of all these arrows that are

being shown here so here's how it works

then in time with multiple pitches so if

you get a bubble opening up cut it in

half now we're looking at a fork so

here's the three prime end of the red

which means the primer is 5 Prime to 3

Prime as it's labeled in blue here so it

starts growing in the left to right

direction

continuously okay so leading strand

this primer here as it's up here grows

from right to left okay but this Fork is

opening so we have double stranded here

this bubble is going to open and we're

going to look at this next figure you're

going to see that so now it's longer

that top strand no problem it keeps

growing right leading strand but this

strand started here now the cell has to

make another primer make another strand

another leg strand which bumps into this

one and now you have okazaki fragments

as that fork opens again it's got to

make another primer make another strand

which will bump into here so now you

have all this discontinuous pieces of

DNA and that needs to be then fixed all

right and so that's what we'll talk

about next here all right so talking

about how to get rid of those primers

and drain those okazaki fragments so DNA

plase 1 then removes the RNA primer with

its exonuclease activity from each of

those okazaki fr ments and replaces it

with DNA all right so it's able to chew

away the RNA and then synthesize new DNA

and it can do this because there are

pre-existing strands right next to it so

if we look here so if it chews away the

green it can use the end of this purple

strand to grow to there choose away this

green it can start synthesizing the end

of that strand up to there so that's how

it's able to um get rid of the RNA and

then to grow the DNA however okazaki

fragments are not coal Bond bonded to

each other after DNA polymerase is done

so there needs to be an additional

enzyme that creates that last

phosphoester bond between those okazaki

fragments and that's a job of an enzyme

called DNA ligase so the okazaki

fragments are then joined and sealed

together by DNA ligase this joins the

three prime hydroxy on the one strand to

the five Prime phosphate group on the

other strand forming a phosphoester bond

or a complete phoso diester Bond

so what does that look like so again

replication bubble Forks growing you get

these okazaki fragments you got to get

rid of these red primers and you have to

join them

together just yet

another bubble showing the same sorts of

things here and so here now if we look

we get the DNA growing in see the RNA

there DNA continue to grow in then you

get an endonuclease which will remove

parts of the RNA and continue to remove

it and then you get you can barely see

this but DNA Li seals the two fragments

together so once you get rid of all this

red you get sealed by okag fragments so

here's a little bit better picture

showing that so DNA plase 3 synthesizes

it all DNA plase 1 replaces the RNA

primary with DNA DNA liase joins your

DNA plase 1 goes up DNA sorry DNA plas 3

goes through DNA plas 1 gets rid of this

red and then DNA liase you see there's a

little bit of opening there DNA liase

seals those two things together what

does this actually look like what is DNA

liase actually doing um so at the end of

one strand so on this strand this strand

here had the primer connected right

through here so DNA polymerase one came

in got rid of this primer and then it

grew this strand up and it bumped into

it but this hydroxy and this oxygen

group are not coal bonded okay so

there's what's called a Nick in DNA is

it's not completely bonded together

because it's missing just this one

calent Bond so DNA liase comes in needs

some energy uses energy usually from ATP

and it creates then that phosphoester

Bond here together with this side and

this side becomes a phospho diester bond

so that's the job of oops sorry about

that that's the job of DNA liase is to

um link those okaz fragments together by

forming those phosphoester bonds um this

showed you already don't need to look at

that one again all right so that was

mainly procaryotic DNA replication

eukariotic DNA replication is very

similar to procaryotic um DNA

replication with just a few differences

there's going to be more players more

factors involved in ukar DNA replication

they're going to have slightly different

names um but very similar so in UK

carots initiation of DNA replication

begins again at an or origin recognition

complex or orc and there's going to

multiple of those okay where there's

only one in bacterial cells and it uses

proteins called cd6 and

cdt1 and these are helicases okay just

we'll just remember them as helicases

you don't have to remember these

specific words uh UK carat also have uh

numerous DNA plases 15 or more of them

and they have all different names but

they're going to be doing the same

things that the ones in um bacterial

cells do um replication is slower okay

so it does not go as fast as it does in

um procaryotes okazaki fragments are

smaller I said they're 1 to 2,000 base

pairs in procaryotes they about 135

nucleotides long in U ukar um the other

thing that ukar have are these

nucleosomes we talked about chromatin

structure and all this protein these

histone proteins within the cells to

wrap the DNA up in so they need to make

all of those nucleosomes to replace them

they also have to get them out of the

way in order for DNA replication to

occur and um these uh nucleosomes or

these hisone proteins need to be

synthesized and then brought back into

the nucleus so that they can be put back

together with the new DNA that's put

together um

there's enzyme protein chromatin

assembly Factor one or calf one that's

uh thought to play a role in targeting

histones to the replication fork so

getting histones to the right spot so

that they can get put back together with

the DNA that has um has been made so

example here origin replication

recognized by cd6

cdt1 will serve as a helicase it'll open

things up so that replication can begin

um the other thing in UK carots you

probably remember from basic biology is

that replication takes place during the

S phase of the cell cycle so mitosis

cells divide they go into a gap one

phase S phase they duplicate their DNA

and then you go go through a gap two

phase where they have duplicated DNA

before they hit mphase or mitosis and

the cell division

phase um so here's just another picture

showing Centere multiple Origins oops

multiple or origins of replication so

opening opening opening opening and

those origins of replication then will

just run into one another so you get

duplicated chromosomes and again um

they're going to run into one another

that will produce Nick or lack of a

coent bond and DNA liase again is going

to be needed to uh join those uh

discontinuous pieces of

DNA all right so there's a number of

tables here that um are that I'll show

you here and um in DNA replication for

eoli we went over DNA a um we talked

about the Heil case DNA B topias single

stranded primas DNA plases um we don't

go over the twos or the T us so worry

about that but you should be able to uh

know except for DNA C except for DNA C

and tus you should be able ble to um

understand what all these different

enzymes are doing during this process um

from eoli bunch of different subunits

don't worry about it and we're not going

to worry about it we'll just know that

there's three different DNA

plases um mutants just tells you what

happens um it's not a bad idea to sort

of understand if something doesn't

happen what um type of subunit must have

not been working and then eukariotic DNA

ples as I mentioned there's more of them

um again they get the stuff done they

just break it down in or numerous ones

like these two here replication of

leading strand versus sling strand

damaged DNA um replication with Prim a

so there's just more of them and a lot

of them involved in DNA repair don't

worry about knowing any of these names

just know UK carots have more DNA plases

doing um this job okay so the other

unique thing then in ukar is you have

tiir or the end of chromosomes so

there's a special way in which that T

DNA is replicated um because DNA plasis

cannot initiate new DNA synthesis when

an RNA primer is removed from the five

Prim and a gap is present so at the end

of DNA there's a problem there is a gap

that is present when DNA pulas 3 comes

in and gets rid of that RNA primer if

this is not corrected chromosomes would

get shorter and shorter every cell cycle

which is thought to be a mechanism by

which some cell

age right and we know this also because

uh immortalized cells or tumor cells

oftentimes have an increase in tase

activity so tase is the enzyme we're

going to talk about here that solves the

problem of um making the ends of DNA and

so this isn't happening in all cells all

the time but it certainly is happening

in our germ cells or cells that make egg

and sperm and it also happens happens a

lot in stem cells cells that um serve as

precursors to all the cells in our body

so an enzyme tase maintains the

chromosomal lengths by adding tiir

repeats to the chromosome ends in some

but not all cells work done on a

protozone shows how this works and we'll

talk about this so tase is made of made

up of made up of both RNA and protein

and this serves um or we'll see how this

works in in its function the RNA

component is complementary to the tiir

repeat unit in the DNA through base

pairing and an overhang tarase will

extend one strand of the DNA in the 5

to3 Prime Direction okay after extending

the Strand it serves as a template for

the plase and that plase and tase leaves

the RNA primary leaves and the RNA

primase makes a new RNA primer that DNA

polymerase uses to fill the gap on the

other strand the new primer is still

removed creating another Gap but the

overall length the DNA strand has been

lengthened now that doesn't make a whole

lot of sense uh talking about it so

we'll walk through the process with a

couple of images all right first here

shows what the problem is so T tiic

repeat sequences you can see these

repeats of these same sequences um there

was an RNA primer here all right so the

five Prime grew to the three prime DNA

pulas came in took it out but it needs

another primer out to the right here to

fill in well because if it's an end of a

DNA that primer doesn't exist and so now

you have this piece and eventually it

get shorter and shorter and shorter and

shorter every time the cell divides so

you need something like Tom Ras to fix

that so again here shows the same thing

DNA ply cannot link the nucleotides on

this end because there is no primer

there's no place for a

primer all right so here's the problem

again you got this Gap here so what

happens so here's tasin again it's made

up of an enzyme and an RNA and so here's

the RNA here that RNA is complementary

to this end of the longer okay not the

one that's been removed but the longer

strand on the DNA and so it hydrogen

base pairs and then tase enzyme part

adds

nucleotides here all right so it uses

this RNA as the template to add the DNA

and so it can shift down and continue to

do that till it gets longer and longer

what then happens is the natural

replication takes place in RNA prime

prime will come in make an RNA primer

DNA plase 1 will go it'll fill in here

DNA plase 3 will come in and it will

chew away this RNA primer again so this

strand on top will still be longer than

the Strand on bottom but if you look at

where the Strand on the bottom ended

earlier it's way back here now it's been

extended way out to here almost to the

end the original end of that top strand

so here's another figure here showing

same thing adding tiles so t is here

it's base pairing which allows this

strand to grow on top which allows Prim

a to come in create another primer fill

in this way all the way down to there

DNA plary 3 will come in and chew that

away but again it's extended it all the

way up to this point so the light blue

here all the way up to here extended it

from here to here and it'll do that

however many times it needs to to get it

to the right length so that's how taras

Works serves it hydrogen base pairs

serves as a template to grow the top

strand in a five Prime and three prime

Direction which then allows primes to

come in create a new primer DNA plase 1

to grow DNA plase 3 removes that and

again I'm saying DNA plase 1 DNA plas 3

those are actually procaryotic enzymes

but the functions of those that are

operating in UK carots and so this then

concludes uh the four parts on DNA