Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 14

STRUCTURE OF TRIPLE-STRANDED DNA

473

Figure 14.5

Acid-stabilized triplex base pairing schemes. A dash (-) indicates the normal Watson-

Crick base pairing scheme while a colon (:) indicates the base pairs which involve the third strand.

(a) As written schematically.

(b) Actual proposed structures.

tiparallel to each other; the Watson – Crick pyrimidine strand is antiparallel to the

purine

strand; the triplex pyrimidine strand is parallel to the purine strand. The specific structural

models proposed require that the pyrimidine sequences have mirror symmetry. This can

be tested by manipulating particular DNA sequences, and it turns out to be valid. In addi-

tion the two models in Figure 14.4 can be evaluated by looking at the pattern of accessi-

bility of the S1 hypersensitive structure to various agents that chemically modify DNA in

a structure-dependent manner. These studies reveal that the correct model for the S1 hy-

persensitive structure is the one shown on the right in Figure 14.4, where the 3

segment

of the purine stretch is the one incorporated into the triplex. The reason why this structure

tematically. The

need for superhelical density to drive the formation of triplex can

be

avoided simply by working at a low enough pH. In practice, pH 5 to 6 suffices for most

sequences capable of forming triplexes at all. A surprise was the remarkable stability of

triplexes. They

can survive electrophoresis, even with lengths as small as 12. Proof

that

pyrimidine,

strand

is antiparallel to the Watson – Crick pyridine strand, was obtained by

an elegant

series

of experiments in which agents were attached to the ends of the third

strand that

were capable of chemically nicking bases on the duplex. The specific pattern

of nicks provided a detailed picture of the structure of the complex (Fig. 14.6).

A second type

of DNA triplex, stable at pH 7 was soon rediscovered. This purine-

purine-pyrimidine

(RRY) was precisely the form known two decades before, stabilized

of the third strand were used

to help analyze the structure

of the

triplex. The appeal of

this approach is that it will be relatively easy to find or introduce a unique DNA sequence

capable of forming triple strands into a target of interest. Subsequent cleavage at this se-

quence would represent the sort of cut that is extremely useful for executing any of the

Smith-Birnstiel-like mapping strategies we described in Chapter 8.

Chemical cleavage agents that have been tried include Cu-phenanthroline complexes,

iron-EDTA-ethidium bromide complexes, and others shown elsewhere in the chapter. The

types of reactions one would like to be able to carry out with these modified oligonu-

cleotides

are shown schematically in Figure 14.10. Rather good yields and specificities

have

been

observed when the chemical cleavage is

used to cut the complementary strand

of a duplex (Fig. 14.10

a ). Much less success has been had with direct triplex-mediated

cleavage

of a duplex (Fig. 14.10

b ). Generally, nicking of one strand of the duplex pro-

ceeds very well, but it is difficult to make the second cut needed to affect a true double-

strand cleavage. The reason for this is that many of the chemical agents used are stoichio-

metric rather than catalytic. They have to be reactivated or replaced by a fresh reagent in

order

to

be able to perform a second strand cleavage. While elegant

chemical

methods

have been proposed to circumvent this problem,

to date, specific efficient duplex chemi-

cal cleavage has been an elusive goal. However, this has not proved to be a serious road-

block, because alternative methods for using

triplexes to

promote

specific

enzymatic

cleavage of duplexes have been very successful.

Achilles’s heel strategies are based on the general notion of using restriction methyl-

ases to protect all except a single or small set of protected recognition sites. This renders

most

of

the potential sites in the sample resistant to the conjugate restriction nuclease.

Then

the

protecting agent is

removed, and the

nuclease added. Cleavage only occurs at

tive group

(x) that generates radicals.

(a) Cleavage of a single strand.

(b) Cleavage of a duplex.

TRIPLEX-MEDIATED DNA CLEAVAGE

477

the sites that escaped the initial protection. These strategies were named by one of their

developers, Wacslaw Szbalski, at the University of Wisconsin, by analogy to the myth of

Achilles. As an infant Achilles was dipped into the river Styx by his mother which ren-

dered him immune to all physical harm except for his heel, which was masked from the

effects of the Styx because his mother was holding him there. Two straightforward exam-

ples of Achilles’s heel specific cleavage of DNA are shown in Figure 14.11. These ap-

proaches are applicable to very large DNA

or even intact genomic DNA, since they can

be carried out in situ in agarose. In one

case it is necessary to find a tight binding site for

a protein that masks a restriction site

(Fig.

14.11

a ).

Examples of suitable protein and

binding sites are lac repressor, lambda repressor,

E. coli

lexA protein, or a host of eukary-

otic

transcription factors,

particularly

viral factors

like

the NFAT protein. To be

useful,

the site must contain an internal or nearby flanking restriction site. There is no guarantee

that such a site will conveniently exist

in a target of interest. However, given the large

number of potentially useful restriction

sites, there are many possibilities. If necessary,

for some applications the desired site can always be designed and introduced.

Instead of proteins, triplexes can

be used to mask restriction sites (Fig.

14.11

b ). It

turns

out that embedding a

four-base restriction

site

in

a homopurine-homopyrimidine

Figure 14.11

Achilles’s heel strategies for specific DNA cleavage.

(a) Blocking a restriction

en-

zyme cleavage site

E with a DNA binding protein.

(b) Blocking a restriction enzyme cleavage site

E

with a triplex.

M indicates methylation sites.

478

SEQUENCE-SPECIFIC MANIPULATION OF DNA

stretch destabilizes the resulting triplex only slightly. However, triplex formation renders

the site totally unaccessible to the restriction methylase. After the remaining restriction

sites have been methylated, conditions are altered to dissociate the triplex. Then the re-

striction enzyme is added, and cleavage is allowed to occur. This Achilles’s heel approach

works very well, even at the level of single sites in the human genome. However, it still

suffers from the limitation that only a small subset of sequences within a target will be

potential sites of triplex-mediated specific cleavage.

A

generalization of the Achilles’s heel approach is

possible by the use of the

E. coli

recA protein. Developed by Camerini-Otero and elaborated by Szybalski (Koob et al.,

1992), this method has been called recA-assisted restriction endonuclease (RARE) cleav-

age (Fig. 14.12). The method is applicable to genomic DNA because all of the steps can

be carried out in agarose. The recA protein has a number of different activities. One of

these is a cooperative binding to single-stranded DNA, leading to a completely coated

complex containing about one recA monomer for every five

bases

(Fig.

14.12

a ). The

coated complex will then interact with double-stranded DNA molecules in a search for

sequences homologous to the single strand. In

E. coli

this process constitutes one of the

early steps that eventually leads to strand invasion and recombination. In the test tube,

without

accessory

nucleases,

the

reaction stops

if

a

homologous

duplex

sequence

is

found,

and the third strand remains complexed to this

homolog,

even

if

the recA

protein

is subsequently removed (Fig. 14.12

b ). The mechanism of the sequence search is un-

known. Similarly the actual nature of the complexes formed with recA protein present or

after

recA protein removal are still not completely understood despite intense efforts to

study

these processes because of their importance in basic

E. coli

biology. Some sort of

triple strand is believed to be involved, although this has never been proven. What is key,

however,

for

Achilles’s

heel

applications,

is

that

the recA

protein-mediated

complex

blocks

the access of restriction methylases to duplex DNA sequences contained within it.

A

schematic outline of RARE cleavage is given in

Figure

14.12

c . The technique has

worked well to cut at two selected sites 200 to 500 kb apart in a target to generate a spe-

cific internal fragment, and generation of a 1.3-Mb telomeric DNA fragment that requires

only a single RARE cleavage has been reported. In practice, it has been more efficient to

use a six-base specific restriction system like rather than the four-base systems used with

other

Achilles’s heel methods. The reason is that a common

source

of

background

in

these approaches is incomplete methylation. This produces a diverse distribution of hemi-

methylated sites which are cut, albeit slowly, by the conjugate nuclease. The result is a

significant background of nonspecific cleavage. This background

can be markedly re-

duced by going to the six-base enzyme, since its sites are 16 times less frequent, on aver-

age. Since recA-mediated cleavage is applicable, in principle, to any selected DNA se-

quence, the rarity of six-base cleavage sites does not pose a particular obstacle.

The recA protein-coated single strands can be as short as 15 bases for RARE cleavage,

although

in

practice

targets

two to four times this length

are

usually

employed.

One

makes a trade-off between the increased efficiency and specificity obtained with longer

complexes, and the lowered efficiency of their diffusion into agarose-embedded DNA

samples. Yields of the desired duplex of 40% to 60% have been reported in early experi-

ments.

It remains

to

be

seen

how

generally

obtainable

such high yields will be. The

power

of

RARE

cleavage in

physical mapping

is

that

given

two

DNA

probes

spaced