dna technology lect 3

8/12/2019 DNA Technology Lect 3

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The overal l repl ication proc ess and synthesis o f

new DNA strands catalyzed by DNA polym erase


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DNA polymerases are processive enzymes

Processivity is a characteristic of enzymes that operate on polymeric

substrates.

In the case of DNA polymerases, the degree of processivity is defined

as the average number of nucleotides added each time the enzyme

binds a primer:template junction.

Each DNA polymerase has a characteristic processivity that can range

from only a few nucleotides to more than 50,000 bases added per

binding event.

It is the initial binding of polymerase to the primer:template junction

that is the rate-limiting step.


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A completely non-processive DNA polymerase would addapproximately 1 base/second.

In contrast, the fastest DNA polymerases add as many as 1,000

base/second by remaining associated with the template for multiple

rounds of dNTP addition.

Increased processivity is facilitated by sequence-independent,

electrostatic interactions between:

a- the phosphate backbone and the thumb domain

b- the minor groove of DNA and the palm domain


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DNA polym erases synthesize DNA

in a processive manner


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Further increases in processivity are achieved through interactions

between the DNA polymerase and a sliding clamp protein that

completely encircles the DNA.

These proteins are composed of multiple identical subunits that

assemble in the shape of a doughnut.

The three dimensional structure of s l id ing DNA clampls

Sliding

clamp

from

E.coli,

a dimer

Sliding

clamp

from

eukaryot ic

cells,a tr im er


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In the presence of the sliding clamp, the DNA polymerase still

disengages its active site from the 3OH end of the DNA frequently.

In the absence of the sl id ing clamp , a DNA po lymerase

diss ociates and d if fuses away from the template DNA on

average once every 20-100 base pairs s yn thes ized.

Bu t the association w i th the sl id ing clamp p revents thepolymerase from di f fusing away from the DNA.

How does the association with the sliding clamp change the

processivity of the DNA polymerase?


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The complex between the DNA sl id ing clamp and

the DNA polymerase:dsDNA com plex

By keeping the DNA polymerase in close proximity to the DNA, the

sliding clamp ensures that the DNA polymerase rapidly rebinds the

same primer:template junction, vastly increasing the processivity of theDNA polymerase.


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Sliding clamp loaders are five subunit protein complexes whose activity

is controlled by ATP binding and hydrolysis.

In E.colithe clamp loader is called the - complexand in eukaryotic cells

is called replication factor C(RFC).

To catalyze the sliding clamp opening, the clamp loader must be bound

to ATP, once bound to ATP, the clamp loader binds the sliding clamp and

opens the ring at one of the subunit:subunit interfaces.

The resulting complex can now bind to DNA at the primer:template junction.

Because only an ATP-bound clamp loader can bind to the clamp and

to DNA, the ADP form of the clamp loader rapidly dissociates from theclamp and the DNA.

This leaves a closed clamp positioned around the dsDNA portion of the

primer:template junction.


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ATP con trol of s l id ing DNA clamp loading

Clamp loader

ATP binds to clamp loader

ATP-clamp loader opens

the sliding clamp

ATP-clamp loader loading the opened sliding

clamp around template:primer junction

ADP-clamp loader dissociates from the

sliding clamp-dsDNA complex

ATP hydrolysis to ADP and releases high energy


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Sliding DNA clamps increase the process ivi ty of DNA po lymerases


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Exonucleases proofread newly synthesized DNA

A system based only on base-pair geometry and thecomplementarity between the bases is incapable of reaching the

extraordinarily high levels of accuracy that are observed for

DNA synthesis in the cell (approxim ately 1 m istake in every

1010base pairs added).

A major limit to DNA polymerase accuracy is the occasional

(approximately once in 105times) "wrong" tautomeric form of

the (imino or enol).

These alternate forms of the bases allow incorrect base pairs

to be correctly positioned for catalysis. Proofreading allows

these mistakes to be corrected.


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Proofreading of DNA synthesis is mediated by nucleases (proofreading

exonucleases)that remove incorrectly base-paired nucleotides.

These exonucleases are capable of degrading DNA starting from a 3'

DNA end, that is from the growing endof the new DNA strand.

Nucleases that can only degrade from a DNA end are called

exonucleases; nucleases that can cut in the middle of a DNA strand arecalled endonucleases.

Thus, in the rare event that an incorrect nucleotide is added to the primer

strand, the proofreading exonuclease removes this nucleotide from the 3'

end of the primer strand.

This "proofreading" of the newly added DNA gives the DNA polymerase a

second chance to add the correct nucleotide.


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Proofr eading exonucleases

removes bases from 3OH

end of mismatched DNA


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The removal of mismatched nucleotides is facilitated by the reduced

ability of DNA polymerase to add a nucleotide adjacent to an incorrectly

base-paired primer.

Mispaired DNA alters the geometry of the 3'-OH and the incoming

nucleotide due to poor interactions with the palm region.

This altered geometry reduces the rate of nucleotide addition in muchthe same way that addition of an incorrectly paired dNTP reduces

catalysis.

Thus, when a mismatched nucleotide is added, it both decreases the

rate of new nucleotide addition and increases the rate of proofreadingexonuclease activity.


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As with DNA synthesis, proofreading can occur without releasing the DNA

from the polymerase.

When a mismatched base pair is detected by the polymerase, the

primer:template junction slides away from the DNA polymerase active site

and into the exonuclease site.

This is because the mismatched DNA has a reduced affinity of the palm

region.

After the incorrect base pair is removed, the correctly paired

primer:template junction slides back into the DNA polymerase active siteand DNA synthesis can continue.


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The special izat ion o f DNA po lymerases

DNA polymerases are specialized for different roles in the cell.

The efficient and accurate replication of the genome requires that cells

have multiple specialized DNA polymerases.

For example, E. coli has at least five DNA polymerases that aredistinguished by their enzymatic properties, subunit composition, and

abundance.

DNA polymerase III (DNA Pol III) is the primary enzyme involved in the

replication of the chromosome.

Because the entire E. coli genome is replicated by two replication

forks, DNA Pol III must be highly processive.


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Consistent with these requirements, DNA Pol III is generally found to

be part of a larger complex that confers very high processivity, a

complex known as the DNA Pol III holoenzyme.

In contrast, DNA polymerase I (DNA Pol I) is specialized for the

removal of the RNA primers that are used to initiate DNA synthesis.

For this reason, this DNA polym erase Ihas a 5' exonucleasethat

allows DNA Pol I to remove RNA or DNA immediately upstreamof thesite of DNA synthesis.

Unlike DNA Pol III, DNA Pol I is not highly processive, adding only

20 100 nucleotides per binding event.

These properties are ideal for RNA primer removal and DNA synthesis

across the resulting ssDNA gap.


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The 5' exonuclease of DNA Pol I can remove the RNA-DNA linkage

that is resistant to RNAse H.

The short extent of synthesis by DNA Pol I is ideal for replacing the

short region previously occupied by the RNA primers (


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Prokaryotic (E. co li) Number of Function

subunits

Pol 1 RNA primer removal, DMA repairPol II 1 DNA repairPol III core 3 Chromosome replicationPol III holoenzyme 9 Chromosome replicationPol IV 1 DNA repairPol V 3 DNA repair


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Eukaryotic cells also have multiple DNA polymerases, with a typical

cell having more than 15 DNA polymerases.

Of these, three are essential to duplicate the genome: DNA Pol d,

DNA Pol e, and DNA Pol a/primase.

Each of these eukaryotic DNA polymerases is composed of multiple

subunits.

DNA Pol a/primase is specifically involved in initiating new DNA

strands. This four-subunit protein complex consists of a two-subunit

DNA Pol aand atwo-subunit primase.

After the primase synthesizes a RNA primer, the resulting RNA primer

:template junction is immediately handed off to the associated DNA Pol

ato initiate DNA synthesis.


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Due to its relatively low processivity, DNA Pol a/primase is rapidly

replaced by the highly processive DNA polymerases d and e.

The process of replacing DNA Pol a/primase with DNA Pol d or eis

called polymerase switchingand results in three different DNA

polymerases functioning at the eukaryotic replication fork.

As in bacterial cells, the majority of the remaining eukaryotic DNA

polymerases are involved in DNA repair.


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Eukaryotic Number of Functionsubunits

Pol a 4 Primer synthesis during DNAreplication

Pol b 1 Base excision repairPol g 3 Mitochondrial DNA replication

and repairPol d 2-3 DNA replication; nucleotide

and base excision repairPol 4 DNA replication; nucleotide and

base excision repair

Examples of eukaryot ic DNA polym erases


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DNA polymerase swi tch ing dur ing eukaryot ic DNA repl icat ion


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Linking number is an invariant topological property of

covalently closed, circular DNA (cccDNA)

Because there are no interruptions in either polynucleotide chain,the two strands of cccDNA cannot be separated from each other

without the breaking of a covalent bond.

If we wished to separate the two circular strands without

permanently breaking any bonds in the sugar-phosphate backbones,we would have to pass one strand through the other strand

repeatedly.

The number of times one strand would have to be passed through

the other strand in order for the two strands to be entirely separatedfrom each other is called the l ink ing number.


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Topolog ical states o f co valent ly clos ed, circu lar DNA (ccc DNA)

Conversion of the relaxed DNA (a) to the negatively supercoiled (b)

form of DNA by the action of topoisomerase


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Topoisomerases

During replication, the unwinding of DNA may cause the formation of

tangling structures, such as supercoils. The major role oftopoisomerases is to prevent DNA tangling.

Structu re of the Topo I/DNA complex

dsDNA Topoisomerase I
http://www.web-books.com/MoBio/Free/Ch7D.htmhttp://www.web-books.com/MoBio/Free/Ch7D.htm


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Types of topoisomerases

There are two types of topoisomerases:

Type I produces transient single-strandbreaks in DNA.

Type II produces transient double-strandbreaks.

The type I enzyme removes supercoils from DNA one at a time.

The type II enzyme removes supercoils two at a time.

Although the type II topoisomerase is more efficient in removing

supercoils, this enzyme requires the energy from ATP hydrolysis, but the

type I topoisomerase does not.


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The topo I of both prokaryotes and eukaryotes are the type I

topoisomerase.

The eukaryotic topo II, bacterial gyrase, and bacterial topo IV belong tothe type II.

In eukaryotes, the topo I and topo II can remove both positive and

negative supercoils.

In bacteria, the topo I can remove only negative supercoils.

The bacterial topo II is also called the gyrase, which has two functions:

(a) to remove the positive supercoils during DNA replication,

(b) to introduce negative supercoils (one supercoil for 15-20 turns

of the DNA helix) so that the DNA molecule can be packed into

the cell. During replication, these negative supercoils are

removed by topo I.


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The bacterial topo IV belongs to the type II topoisomerase. This enzyme

is involved in decatenation.

During replication, these negative supercoils are removed by topo I.

The bacterial topo IV belongs to the type II topoisomerase. This enzyme

is involved in decatenation.

Without topoisomerases, the DNA cannot replicate normally.

Therefore, the inhibitors of topoisomerases have been used as anti-

cancer drugs to stop the proliferation of malignant cells.

However, these inhibitors may also stop the division of normal

cells. Some cells (e.g., hair cells) which need to continuously divide will

be most affected. This explains a noticeable side effect: the hair loss.


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Type I topoisomerases can only catenate and

decatenate mo lecules if DNA strand has a nick

or a gap.

This is because these enzymes cleave on ly

one DNA strand at a time


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Type II topo isomerases can catenate and decatenate

cccDNA by introdu cing a doub le-stranded break in

one DNA and passing the other DNA molecule

through the break


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( c) Entangled long l inear DNA molecules, generated

for examp le, du ring the repl icat ion of eukaryot icchromosomes, can be distang led by top oisom erase II.

(d) DNA knots can be unknotted by topoisomerase II.


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DNA topoisomers can be separated by electrophoresis

Covalently closed, circular DNA (cccDNA) molecules of the same length but

of different linking number are called DNA topoisomers.

Even though topoisomers have the same molecular weight, they can be

separated from each other by electrophoresis through a gel of agarose.

The more compact the DNA, the more easily it is able to migrate through

the gel matrix.

Thus, a fully relaxed cccDNA migrates more slowly than a highly supercoiledtopoisomer of the same circular DNA.


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Topoisomerases use a covalent protein-DNA

linkage to cleave and rejoin DNA strands

To perform their functions, topoisomerases must cleave a DNAstrand (or two strands) and then rejoin the cleaved strand (or strands).

Topoisomerases are able to promote both DNA cleavage and

rejoining without the assistance of other proteins or high-energy

co-factors (for example, ATP) because they use a covalent-intermediate mechanism.

DNA cleavage occurs when a tyrosine residuein the active site of

the topoisomerase attacks a phosphodiester bond in the backbone of

the target DNA

This attack generates a break in the DNA, whereby the

topoisomerase is covalently joined to one of the broken ends via a

phospho-tyrosine linkage. The other end of the DNA terminates with a

free OH group.


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The phospho-tyrosine linkage conserves the energy of the

phosphodiester bond that was cleaved.

Therefore, the DNA can be resealed simply by reversing the original

reaction.

The OH group from one broken DNA end attacks the phospho-tyrosine

bond reforming the DNA phosphodiester bond.

This reaction rejoins the DNA strand and releases the topoisomerase,

which can then go on to catalyze another reaction cycle.

Although as noted above, type II topoisomerases require ATP-hydrolysisfor activity, the energy released by this hydrolysis is used to promote

conformational changes in the topoisomerase-DNA complex rather than

to cleave or rejoin DNA.


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Phospho- tyros ine

cov alet in termediate

Cleavage and rejoining o f DNA using a covalent

tyr os ine-DNA intermediate


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1 2 3 4 5 6Kb

9.5

7.5

4.4

2.4

0.24

1- RNA ladder

2 & 3 Partially degrade RNA

5- completely degrade RNA

6- Undegraded total RNA

(good quality RNA)

dna technology lect 3

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