Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 1

DNA

SYNTHESIS

11

BOX 1.2

(Continued)

Exonucleases remove nucleotides from the ends of DNA. Most cleave single nu-

cleotides one at a time off one end of the DNA. For example, Exonuclease III carries

out steps like

5

ApCpGp

——— pTpApA

3

3

TpGpCp

——— pApTpT

5

5

Ap

CpGp ——— pTpA–OH

3

3

HO–GpCp ——— pApTpT

5

5

ApCpGp

——— pT–OH

3

3

HO– Cp

——— pApTpT

5

and continues until there are no double-stranded regions left.

Terminal transferases add nucleotides at the 3

-end of a DNA

strand. They

require

that

this end

not have a 3

-phosphate. No

template is required; hence

the

sequence of

the incorporated residues is random. However, if only a single type of pppdN is used,

then a set of homopolymeric products is produced

GpApTpCpA

pppdT

9

GpApTpCpA:

(pT)

n

tranferase

where

n

is variable. Some DNA polymerases

(Box

1.6) also

have

a terminal

trans-

ferase activity (see Box 1.5).

As shown in Figure 1.6

b,

synthetic DNA duplexes can be strung together, and nicks in the

polynucleotide backbone can be sealed with the enzyme DNA ligase. This enzyme re-

quires a 5

-phosphate group and

a free 3

-OH in order to form a phosphodiester bond

(Box 1.2). There are two commonly used forms of the enzyme: the species isolated from

Escherichia coli

uses NAD as an energy source, while the bacteriophage T7 enzyme re-

quires ATP. Thermostable ligases isolated from thermophilic organisms are also available.

Their utility will be demonstrated later.

Once a duplex DNA is available, it can be immortalized by cloning it into an appropri-

ate vector (see Box 1.3). Duplexes (or single strands) can also be amplified in vitro by

methods such as the polymerase chain reaction (Chapter 4). The result is that for any de-

sired species, a chemical synthesis needs only to be done once. Thereafter biological or

enzymatic methods usually suffice to keep up the supply of product. It remains to be seen

in the future, as larger stocks of specific synthetic DNA sequences accumulate world-

wide, whether it is worthwhile to set up a distribution system to supply the potentially

millions of different compounds to

interested users, or whether it will be simpler and

cheaper to manufacture a needed compound directly on site.

By taking advantage of the specificity of base pairing, it is possible to synthesize and as-

semble more complex DNA structures than those that predominate in nature. Naturally oc-

curring DNAs are

either circles with

no

ends or linear duplexes

with two protected ends.

14

DNA CHEMISTRY AND BIOLOGY

BOX 1.3

(Continued)

and release bacteriophage, then a convenient method of cloning is to grow a continu-

ous layer of susceptible bacteria and then cover the culture with a thin layer of dilute

bacteriophage suspension. Individual bacteriophage will infect cells, leading to contin-

ual cycles of bacteriophage growth and cell lysis until visible holes in the bacterial

lawn, called bacteriophage plaques, are observed. Individual colonies or plaques are

selected for further study by picking, literally by sampling with a sterile toothpick or

other

sharp

object, and then reinnoculated, respectively, into a

permissive growth

medium or strain.

A key variable in the choice of vector is the range of target sizes that can be accom-

modated. Plasmids generally allow a broad range of target sizes, while bacteriophages,

because of constraints in the packaging process, generally accept only a narrow range

of target sizes. The earliest cloning vectors were efficient for relatively small targets,

under 10 kb. Progressively interest has turned to ever larger targets, specialized vec-

tor/host systems needed to propagate such targets are described later (Boxes 2.3 and

8.2).

A second key variable in the choice of cloning vector is the copy number. Some

plasmids and

bacteriophages can grow in tens of thousands of copies per

E. coli

cell.

This

greatly

facilitates recovery and purification of cloned DNA inserts. However, if

DNA fragments toxic to the host are the desired targets, a high copy number vector

system will make it more difficult for them to be propagated.

The major advantages of cloning, as compared to the in vitro replication systems

discussed in Chapter 4, are the ability to handle very large numbers of samples in par-

allel and the absence of a need for any prior knowledge about the desired target se-

quences. The major disadvantages of cloning are that it is a relatively time-consuming

process difficult to automate, and some DNA sequences are not cloneable either be-

cause they are toxic to particular hosts, or they are unstable in those

hosts and frag-

ments rearrange or delete.

tures

are

actually

seen in

cells as intermediate steps in genetic recombination. However,

these

so-called Holliday structures are unstable, since the participating strands are pairs of

identical

sequences. The

location of the junction can move around by

a process

called

branch migration.

Synthetic junctions can be designed that are

stable because their DNA

sequences do not

allow branch migration. At low salt concentrations these structures

be-

shaped structure with one pair of co-axially stacked helices crossing over a

second

pair

of

co-axially stacked helices.

Still other types of DNA structures can be formed by taking advantage of

the

fact

that

in the double helix, specific hydrogen bond donor and acceptor sites on the bases remain

exposed in the major groove of the double helix. Under appropriate conditions this allows

the association of a third DNA strand in a sequence-specific manner

to

form

a

DNA

triplex. It produces a DNA structure with four ends. The great utility of

triplexes,

which

we will exploit in considerable detail later (Chapter 14), is that

triplexes

can

be formed

DNA AS A FLEXIBLE SET OF CHEMICAL REAGENTS

15

able compositions. DNAs

and RNAs are ideal aptamers because powerful methods

exist

to work with very complex mixtures of species and to purify from these mixtures just the

molecules with high affinity for a target from which to characterize the common features

of these classes of molecules. These cycles of design and optimization form

the heart of

any engineering process. (See Chapter 14 for a detailed discussion.)

The goal, in nanoengineering is to make machines, motors, transducers, and

tools at

the molecular level. It is really the ultimate chemical synthesis, since entire arrays of mol-

ecules (or atoms) must be custom designed for specific mechanical or electromagnetic

properties. The potential advantages of using DNAs for such purposes is due to several

factors: the great power to synthesize DNAs of any desired length and sequence, the abil-

ity to make complex twoand three-dimensional arrays using Holliday junctions, and the

formation

of structures

of accurate length by taking advantage of the great stiffness

of

duplex DNA. (See

Box

1.4.) Proteins can be anchored along the DNA at

many

points

to create

arrays

with

more complex properties. The disadvantages of using

DNA

and

Figure 1.9

Use

of DNA molecules

as spacers

to construct long molecular rulers.

(a) Symbols

used.

(b)

Noncovalent

coupling.

(c)

Covalent coupling.

BASIC

DNA

BIOLOGY

19

BASIC

DNA

BIOLOGY

DNA is the essential store of genetic information in the cell. That information must be

duplicated to be passed to daughter cells by the process known as

replication.

It must

be

read out as an RNA copy by the process known as

transcription

so

that this

copy (after

editing in many cases) can be used to direct the synthesis of proteins.

DNA replication is limited by the fact that all of the known enzymes that can faithfully

copy the information encoded in DNA have very restricted properties. Such enzymes are

called

DNA polymerases.

They all require a 3

-ended nucleic acid (or a protein substitut-

ing for this 3

-end) as a primer, and a template strand, which is copied. They cannot initi-

ate synthesis de novo but can

only elongate a strand defined by

a primer (Figure 1.10).

They can, for the most part, copy a DNA strand all of the way to its end. Accurate replica-

tion cannot depend on base pairing specificity alone because the

thermodynamic

differ-

ence between the stability of a single base pair and a mismatch is not very great (Chapter

3). Editing is used to correct any misincorporated bases. Some

of this editing is carried

out by the DNA polymerase itself. A

newly incorporated

mispaired

base can

be excised

by the

3

-exonuclease activity of most DNA polymerases. Some

enzymes also have a 5

-

exonuclease activity that degrades any DNA strands in front of a wave of new synthesis.

This process is called

nick translation.

Other enzymes that lack this

activity will

displace

one strand of a duplex in order to synthesize a new strand. This process is called

strand

displacement.

These enzyme activities are illustrated in Box 1.5.

Special procedures are used to synthesize the primers needed to start DNA replication.

For example, RNA primers can be used and then degraded to result in a complete DNA

strand. Both strands of DNA must be replicated. The unidirectional mode of DNA poly-

merases makes this process complicated, as shown in Figure 1.11. In a single-replication

fork the synthesis of one strand (the leading strand), once initiated, can proceed in an un-

interrupted fashion. The other strand (the lagging strand) must be made in a retrograde

fashion from periodically spaced primers, and the resulting fragments are then stitched

together. Most replication processes employ not a single fork but a bidirectional pair of

replication forks, as shown in Figure 1.11.

Any DNA replication process poses an interesting topological problem, whether it is

carried out by a cell or in the laboratory. Because the two DNA strands are wound about a

common axis, the helix axis, they are actually also twisted around each other. They cannot

be unwound without rotation of

the structure, once for each helix turn. Thus

DNA

must

spin as it is being replicated. In the bacterium

E. coli,

for example, the rate of replication

is fast enough to make a complete copy of the chromosomal

DNA in about 40 minutes.

Since the chromosome contains almost 5 million base pairs of DNA, the required replica-

tion rate is 1.2

105

base pairs per minute. Since

E. coli

uses a pair of bidirectional replica-

tion forks, each must move at

6

104

bases

per minute. Thus each side of the replication

fork

must

unwind

6

103 helical

turns

per

minute:

it

must

rotate

at 6

103

rpm.