DNA Replication

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DNA Replication

DNA REPLICATION

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Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


1. DNA Replication

In both prokaryotes and eukaryotes, DNA replication

occurs as a prelude to cell division. This DNA replication

phase is called the S (synthesis) phase. . The two daughter

DNA molecules formed from replication eventually

become chromosomes in their own right in the daughter

cells.

As with all phenomena that involve nucleic acids, the

basic machinery of DNA replication depends on

complementarity of DNA molecules and on the ability of

proteins to form specific interactions with DNA of specific

sequences.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


2. The model of Watson and Crick

The model of DNA replication proposed by Watson and

Crick is based on the hydrogen-bonded specificity of the

base pairs. Complementary strands are shown in different

colors. The fact that new strands can grow only in the

5’-to-3’ direction adds complexities to the detailed

mechanism of replication.

If this model is correct, then each daughter molecule

should contain one parental nucleotide chain and one

newly synthesized nucleotide chain. This prediction has

been tested in both prokaryotes and eukaryotes. A little

thought shows that there are at least three different ways

in which a parental DNA molecule might be related to the

daughter molecules. These hypothetical modes are called

semiconservative (the Watson-Crick model), conservative,

and dispersive

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


3. Three alternative patterns for DNA replication

In semiconservative replication,

each daughter duplex contains one

parental and one newly synthesized

strand. However, in conservative

replication, one daughter duplex

consists of two newly synthesized

strands, and the parent duplex is

conserved. Dispersive replication

results in daughter duplexes that

consist of strands containing only

segments of parental DNA and

newly synthesized DNA

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


4. The Meselson-Stahl experiment


In 1958, Matthew Meselson and Franklin Stahl set out to distinguish among the

three models. . They grew E. coli cells in a medium containing the heavy isotope of

nitrogen 15 N rather than the normal light ( 14 N) form. This isotope was inserted into

the nitrogen bases, which then were incorporated into newly synthesized DNA

strands.


After many cell divisions in 15 N, the

DNA of the cells were well labeled

with the heavy isotope. The cells were

then removed from the 15 N medium

and put into a 14 N medium; after one

and two cell divisions, , samples were

taken. DNA was extracted from the

cells in each of these samples and put

into a solution of cesium chloride

(CsCl) in an ultracentrifuge.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


5. Centrifugation of DNA

in a cesium chloride (CsCl) gradient

If cesium chloride is spun in a centrifuge at tremendously high speeds (50,000 rpm)

for many hours, the cesium and chloride ions tend to be pushed by centrifugal force

toward the bottom of the tube. Ultimately, a gradient of Cs+ and Cl ions is

established in the tube, with the highest ion concentration at the bottom.

Molecules of DNA in the solution also are pushed toward the bottom by centrifugal

force. But, as they travel down the tube, they encounter the increasing salt

concentration, which tends to push them back up owing to the buoyancy of DNA

(its tendency to float). Thus, the DNA finally "settles" at some point in the tube

where the centrifugal forces just balance the buoyancy of the molecules in the

cesium chloride gradient.

The buoyancy of DNA depends on its density (which in turn depends on the ratio of

GC to AT base pairs). The presence of the heavier isotope of nitrogen changes the

buoyant density of DNA. The DNA extracted from cells grown for several

generations on 15 N medium can be readily distinguished from the DNA of cells

grown on 14 N medium by the equilibrium position reached in a cesium chloride

gradient. . Such samples are commonly called heavy and light DNA, respectively.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


6. The proof of the semiconservative model

Meselson and Stahl found that, one generation

after the heavy cells were moved to 14 N

medium, the DNA formed a single band of an

intermediate density between the densities of the

heavy and light controls. After two generations

in 14 N medium, the DNA formed two bands: one

at the intermediate position, the other at the light

position.

This result would be expected from the

semiconservative mode of replication; in fact,

the result is compatible with only this mode if

the experiment begins with chromosomes

composed of individual double helices

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


7. Harlequin chromosomes


With the use of a more modern staining technique, it is now possible to visualize the

semiconservative replication of chromosomes at mitosis. In this procedure, the

chromosomes go through two rounds of replication in the presence of

bromodeoxyuridine (BUdR), which replaces thymidine in the newly synthesized

DNA. The chromosomes are then stained with Giemsa stain, producing the

appearance shown. (The light blue lines represent the BUdR-substituted strands.)

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


8. Visualizing sister chromatids

If cells dividing in culture are treated with

BrdU during S phase, the cells are fooled

into incorporating it — instead of

thymidine — into their DNA.

One of the properties of the resulting

DNA is that it fails to take up stain in a

normal way.

When cells are allowed to duplicate their

chromosomes once in BrdU, the

chromosome that appear at the next

metaphase stain normally.

However, when the cells duplicate their

chromosomes a second time in BrdU, one

of the sister chromatids that appears at

the next metaphase stains normally, while

its sister chromatid does not.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


9. DNA polymerases

In the late 1950s, Arthur

Kornberg successfully

identified and purified the first

DNA polymerase, an enzyme

that catalyzes the replication

reaction.

This reaction works only with

the triphosphate forms of the

nucleotides (such as

deoxyadenosine triphosphate,

or dATP).

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


10. DNA polymerases in E. coli



We now know that there are three DNA polymerases in E. coli. The

first enzyme that Kornberg purified is called DNA polymerase I or

pol I. This enzyme has three activities, which appear to be located in

different parts of the molecule:


1. a polymerase activity, which catalyzes chain growth in the 5’→ 5 3’ direction;

2. a 3’→ 3 5’ exonuclease activity, which removes mismatched bases; and

3. a 5’ 5 → 3’ exonuclease activity, which degrades double-stranded DNA.

Subsequently, two additional polymerases, pol II and pol III, , were

identified in E. coli. Pol II may repair damaged DNA. Pol III, together

with pol I, has a role in the replication of E. coli DNA

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


11. DNA replication fork

The complete complex, or holoenzyme, of pol III contains at least 20 different

polypeptide subunits, , although the catalytic "core" consists of only three subunits.

The pol III complex will complete the replication of single-stranded DNA if there is

at least a short segment of duplex already present.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


12. Prokaryotic origins of replication


E. coli replication begins from a fixed origin, termed oriC, but then proceeds

bidirectionally (with moving forks at both ends of the replicating piece). It is 245 bp

long and has several components. First, there is a side-by-side, or tandem, set of 13-

bp sequences, which are nearly identical. . There is also a set of binding sites for a

protein, the DnaA protein. . An initial step in DNA synthesis is the unwinding of the

DNA at the origin in response to binding of the DnaA protein.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


13. A replicating E. coli chromosome

The DNA has been labeled with 3H-deoxythymidine, and

the radioactivity has been detected by overlaying the

replicating chromosome with photographic emulsion. The

autoradiograph shows that the E. coli chromosome has

two replication forks.

Although there seem to be

two bubbles of replication,

actually the point where the

two smaller bubbles meet is

actually just where two

strands of DNA are laying

across one another

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


14. Eukaryotic origins of replication

Bacteria such as E. coli usually require a 40-minute

replication-division cycle, but, in eukaryotes, the cycle can

vary from 1.4 hours in yeast to 24 hours in cultured animal

cells and may last from 100 to 200 hours in some cells.

Eukaryotes have to solve the problem of coordinating the

replication of more than one chromosome, as well as

replicating the complex structure of the chromosome itself.

In eukaryotes, replication proceeds from multiple points of

origin.

Experiments in yeast indicate the existence of about 400

replication origins distributed among the 17 chromosomes,

and in humans there are estimated to be more than 10,000

growing forks

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


15. Replication bubbles in the fruit fly

Electron micrograph of

replicating DNA in the

embryo of the fruit fly

D. melanogaster

At least 20 different bubbles, therefore with at least 40 different replication forks, can

be observed in this electron micrograph (and accompanying drawn representation of

the electron micrograph.) The large number of replication origins in eukaryotic

chromosomes vs. E. coli's one, enables the slower replication apparatus to copy the

larger eukaryotic genome in approximately the same amount of time as the prokaryotic

genome is replicated

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


16. Replication bubbles

Electron micrograph of

DNA extracted from

rapidly dividing nuclei

of early D. Melanogaster

embryos. The arrows

mark replication

bubbles; the diameters of

DNA chain in both arms

of these bubbles indicate

that they are double-

stranded.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


17. Priming DNA synthesis

DNA polymerases can extend a chain but cannot

start a chain. . Therefore, DNA synthesis must first

be initiated with a primer, , or short oligonucleotide,

that generates a segment of duplex DNA.

RNA primers are synthesized either by RNA

polymerase or by an enzyme termed primase.

Primase synthesizes a short (approximately 30 bp

long) stretch of RNA complementary to a specific

region of the chromosome.

The RNA chain is then extended with DNA by

DNA polymerase. E. coli primase forms a complex

with the template DNA, and additional proteins, such

as DnaB, DnaT, Pri A, Pri B, and Pri C. The entire

complex is termed a primosome.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


18. Leading strand and lagging strand

DNA polymerases synthesize new chains only in

the 5’→ 3’ direction and therefore, because of the

antiparallel nature of the DNA molecule, move in a

3’ → 5’ direction on the template strand. The

consequence of this polarity is that while one new

strand, the leading strand, is synthesized

continuously, the other, the lagging strand, must be

synthesized in short, discontinuous segments. The

addition of nucleotides along the template for the

lagging strand must proceed toward the template's

5’ end (because replication always moves along the

template in a 3’ 3 → 5’ direction so that the new

strand can grow 5’ 5 → 3’). Thus, the new strand

must grow in a direction opposite that of the

movement of the replication fork.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


19. Discontinuous synthesis

As fork movement exposes a new section of lagging-strand template, a new lagging-

strand fragment is begun and proceeds away from the fork until it is stopped by the

preceding fragment.

In E. coli, pol III carries out most of the DNA synthesis on both strands, and pol I fills

in the gaps left in the lagging strand, which are then sealed by the enzyme DNA ligase.

DNA ligases join broken pieces of DNA by catalyzing the formation of a

phosphodiester bond between the 5’ 5 phosphate end of a hydrogen-bonded nucleotide

and an adjacent 3’ 3 OH group. It is the only enzyme that can seal DNA chains.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


20. Steps in DNA synthesis

a) The primers for the

discontinuous synthesis on the

lagging strand are synthesized

by primase.

b) The primers are extended by

DNA polymerase to yield DNA

fragments that were first

detected by Reiji Okazaki and

are termed Okazaki

fragments.

c) The 5’ 5 → 3’ exonuclease

activity of pol I removes the

primers and fills in the gaps

with DNA,

d) which are sealed by DNA

ligase.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


21. A comprehensive view of the replication fork

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


22. Other DNA-modifying enzymes




Helicases are enzymes that disrupt the hydrogen bonds that hold the two DNA

strands together in a double helix. Among E. coli helicases are the DnaB protein

and the Rep protein. The Rep protein may help to unwind the double helix ahead of

the polymerase. The unwound DNA is stabilized by the single-stranded binding

(SSB) protein, which binds to the single-stranded DNA and retards reformation of

the duplex.

The action of helicases during DNA replication generates twists in the circular

DNA that need to be removed to allow replication to continue. Circular DNA can be

twisted and coiled, , much like the extra coils that can be introduced into a rubber

band.

This supercoiling can be created or relaxed by enzymes termed topoisomerases.

There are two basic types of isomerases. Type I enzymes induce a single-stranded

break into the DNA duplex. Type II enzymes cause a break in both strands. . In E.

coli, , topo I and topo III are examples of type I enzymes, whereas gyrase is an

example of a type II enzyme.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


23. The action of topoisomerases


Untwisting of the DNA strands to open the replication fork causes

extra twisting at other regions, and the supercoiling releases the strain

of the extra twisting. During replication, gyrase is needed to remove

positive supercoils ahead of the replication fork

Swivel function of topoisomerase during replication. Extra-twisted

(positively supercoiled) regions accumulate ahead of the fork as the

parental strands separate for replication. A topoisomerase is required

to remove these regions, acting as a swivel to allow extensive

replication.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini

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