Download DNA`s repair kit, packaged in the nucleus, includes

by Albert Rosenfeld
DNA's repair kit, packaged in the nucleus, includes
enzymes for each kind of snip and patch job plus the
ability to whip up emergency crews when needed.
n this cancer-conscious era, most Americans are aware that excessive exposure
to sunlight year in and year out puts
them at some small, long-term risk of developing skin cancer. But it would hardly occur
to the average s u n b a t h e r to think about this
in terms of his DNA being damaged—and
perhaps even repaired—by the sun, or to feel
grateful for not being afflicted with a rare
disease, xeroderma oip-rnentosurri, or XP, that
would prevent the repair of damaged DNA.
The victims of XP nearly always get skin
cancer before the age of 20. Sometimes they
get a variety of skin cancers, including the
much feared malignant melanoma. Often
they get cancers by the dozen, even by the
hundred.
If an XP victim were to lie out in the midsummer sun for even an hour or two on a
single day, he would be in danger of having
already set a c a n c e r - i n i t i a t i n g p r o c e s s in
motion. It was James E. Cleaver of the University of California Medical Center in San
Francisco who demonstrated in the late 1960s
that skin cells of XP victims are genetically
defective; they lack the capacity to repair
damage to their DNA—damage caused, for
instance, by e x p o s u r e to the s u n ' s u l t r a violet radiation.
Everyone is subject to ultraviolet-caused
DNA damage. But h e a l t h y cells have ways to
repair the damage. T h i s was demonstrated
first in bacteria in the early 1960s by another
pioneer investigator of DNA repair, Richard B.
Setlow, then of the O a k Ridge National
Laboratory and now at Brookhaven National
Laboratory.
Amazingly, one of the ways to repair ultraviolet-induced damage is simply to shine
ordinary visible light on the stricken cells.
This process has been studied mainly in bacteria, but Setlow's colleague Betsy M. Sutherland has done enough work with h u m a n
cells in tissue culture to be convinced that
DNA repair by photoreactivation, or light
repair, occurs in h u m a n skin cells as well.
T h o u g h this conviction is not yet shared by
all investigators, sunbathers familiar with
this work might well imagine that the sunlight (containing both ultraviolet and visible
radiation) falling on his skin is alternately
(perhaps simultaneously) damaging and healing their DNA. With an XP victim, it is virtually all one-way and irreversible.
Another leading investigator of DNA repair
is Philip C. Hanawalt of Stanford University, who once studied with Richard Setlow.
Hanawalt, with his own student, David Pettijohn, discovered what is called repair replication, one of the critical steps in the repair
process. Hanawalt and his co-workers have
since elucidated repair p a t h w a y s in both
bacteria and h u m a n cells. T o underline the
radical shift in thought that the discovery of
DNA repair has b r o u g h t about, Hanawalt
points to the fact that until very recently
scientists believed that organisms were able
to maintain their genetic continuity only
because of the inherent stability of their
DNA molecules.
T h e implicit assumption was that, in the
event of DNA damage, the cell either died
or survived despite the persisting damage.
The damage might have a negligible effect
if it occurred at an u n i m p o r t a n t gene site; it
could also be the kind of mutation that drives
evolution. This overly simple notion, however, could not long remain tenable.
T h e more scientists observed the effects
of DNA damage—not only by ultraviolet, X-ray
and other ionizing radiation but by a host of
chemical mutagens and carcinogens too—
and the more they u n d e r s t o o d that alterations in the DNA molecule can also occur
spontaneously, the more it became apparent
that the cell must possess some kind of builtin mechanism for repairing DNA. And this
realization has resulted in a burgeoning of
DNA-repair research in the past decade or so.
T h e promise is a better basic understanding
of genetic and e v o l u t i o n a r y processes as
well as of clues and possible therapies in a
number of h u m a n diseases. Also emerging
are new insights into the specific biochemical
relationship between living organisms and
their environments.
How damage occurs
T h e genetic code is m a d e u p of four basic
"letters," or nucleotides, strung together between a helical pair of complementary strands
and creating the appearance of a spiral staircase or rope ladder. Each nucleotide is made
up of a base—the essential genetic l e t t e r attached to sugar and p h o s p h a t e chemical
g r o u p s that join together to form the molecule's backbone.
T h e four bases, DNA's genetic alphabet,
are usually characterized as A (for adenine),
G (for guanine), T (for thymine), and C (for
cytosine). The bases A and G belong to the
M O S A i n . l f l n i i a r v / F p h r n a r v 1QR1
11
normal DNA
A-Adenine
C - Cytosine
T - Thymine
G- Guanine
Normal replication, DNA divides (from
right to left here) between its strands, along
which are strung complementary sequences of
bases: an adenine (A) always facing a
thymine (T); a guanine (G) always facing
a cytosine (c). Upon division, each single
strand directs the construction of a new complement. Then cell division takes place.
Damage. Uitraviolet radiation can cause
two thymines to bind to each other, creating
what is called a thymine dimer, one of a
number of kinds of ills to which DNA is heir.
Effect of damage. In the absence of repair, the
dimer is unable to order adenines for the
complementary strand facing it. Replication
stops at the gap; the cell dies.
chemical family called purines, while T and
C are pyrimidines. In the DNA double helix, a
purine is always found opposite a pyrimidine;
quite specifically, A always goes with T and
G with C. T h u s if a T were to be knocked out
by, say, the impact of a cosmic ray, an "intelligent" genetic apparatus would "know"—
from the presence of the naked A—that the
hole opposite it would need to be filled by a T.
Every healthy cell does appear to possess
just such an a p p a r a t u s . T h e nucleus contains not only DNA but proteins as well. (See
" T h e S t r u c t u r e s that M a k e DNA W o r k , "
Mosaic, Volume 11, N u m b e r 1, and "DNA
Topology: K n o t s N o Sailor Ever K n e w , " in
this issue.) A m o n g the n u m e r o u s types of
protein molecules in cell nuclei are enzymes
of many kinds that represent the cell's atthe-ready a r m a m e n t a r i u m for DNA repair.
W h y would mere exposure to ultraviolet
light be d a m a g i n g to DNA? O n a given DNA
s t r a n d , two T bases—two thymines—are
often found side by side (across from two
adenines, of course). The atoms of each T are
b o u n d to each other in such a way as to
permit their electrons to absorb ultraviolet
Rosenfeld, a veteran science journalist, holds
an adjunct professorship
at the
University
of Texas Medical Branch in Galveston.
19
Mn.QAlP .laniiarw/Cflhrnarw 1001
energy. But that radiation so excites the electrons when it strikes them that they "reach
o u t " to bond with the electrons of the T next
door. Thus they form what is called a thymine
dimer. T h e radiation can also create dimers
between adjacent cytosines or between a
thymine and an adjacent cytosine. (In chemical terms, a single Base is a monomer, two
joined together form a ^imer, and several
would make a polymer.)
Bonds can also
form between purine bases, but not from
ultraviolet exposure.
But why does the formation of a dimer
constitute damage? As Setlow and his wife,
Jane (an eminent scientist in her own right),
demonstrated while they were still at O a k
Ridge, bases in a dimer are too firmly b o u n d
to each other to carry out their replication
tasks. Dimers have been likened to a place
on a zipper where two adjacent teeth have
fused, thus stopping all action.
N o w , how to unfuse the zipper t e e t h unbind the two bound bases? The mere flicking on of a light switch would not appear to
offer much. But as Claud S. Rupertof Johns
H o p k i n s discovered, the cell nucleus contains enzymes that are sensitive to light—
photoreactivation enzymes. These enzymes
come equipped to recognize the distortion
(often referred to as a b u m p , kink, warp, or
wrinkle) on the DNA strand that signifies
damage—the presence of a thymine dimer.
W h e n light activates it, the enzyme fits itself jigsaw-like to the dimer, splitting it back
into a pair of normal monomers again. No
one yet u n d e r s t a n d s the detailed process by
which it achieves this remarkable feat.
Photoreactivation, like so many other phenomena in science, was discovered serendipitously. In the late 1940s, Renato Dulbecco, now at Caltech, and Albert Kelner,
now at Brandeis, were working independently
of each other at Harvard on ultraviolet irradiation of bacterial viruses and bacteria
respectively. Each inadvertently left his cultures exposed to fluorescent lights overnight
and discovered the next day that many more
of the organisms had survived than would
have been the case had the cultures been
left in the dark.
More is k n o w n about ultraviolet damage
such as thymine dimers than about most
other kinds of DNA damage and repair, because
those have turned out to be, so far, the easiest
to study. But there are ways other than photoreactivation to repair ultraviolet damage—
and of course there are numerous other kinds
of damage. Photoreactivation is also called
light repair. M o s t other kinds of DNA repair
are encompassed under the term dark repair,
light activated repair
Light repair. In those cells that are exposed
to light, a photoreactivation enzyme can
fit itself to the thymine dimer...
...snip the dimer open before the damage
has reached the point of replication, thus...
...freeing the two thymine bases to order
complementary adenine bases across from
them. Replication proceeds.
which is much more common; except for
those cells at an organism's surface, most
cells are never exposed to light.
Dark repair
Setlow, along with a colleague at Oak
R i d g e , W . L. Carrier, was the earliest to
d i s c e r n that u l t r a v i o l e t - i n d u c e d t h y m i n e
dimers (in the common intestinal bacterium
Escherichia coli) could also be repaired in
the dark by means of "excision repair." At
about the time Setlow was making his first
daring sorties into the terra incognita of
DNA repair, Robert Painter of the University of California at San Francisco was discovering, in his mammalian cell cultures, a
p h e n o m e n o n he called u n s c h e d u l e d DNA
s y n t h e s i s — t h a t is, s y n t h e s i s taking place
w h e n the cells were not carrying out their
normally programmed replication. W h a t he
was observing, it turned out, was a part of
excision repair.
Also at about the same time, Hanawalt
and his Stanford colleagues identified in
bacteria another excision-repair component—
the insertion step. And by 1968, James D.
Regan and his colleagues at Oak Ridge Nat i o n a l Laboratories were finding excision
repair in h u m a n cells. Since then excision
repair—the kind Cleaver found missing in
the cells of XP victims—has probably been
the most extensively studied type of DNA repair. In excision repair, to put it oversimply,
the dimer or other damaged area is snipped
out and replaced either with a " s h o r t p a t c h "
(three or four nucleotides) or a "long p a t c h "
(as many as a hundred nucleotides), depending on the extent and nature of the damage.
As Setlow and Carrier first described the
process, excision repair occurs in four steps,
requiring the cooperation of at least four
specific enzymes in the cell's arsenal. First,
an enzyme called an endonuclease (the "ase"
ending always denotes an enzyme) recognizes
the site of the damage and cuts a nick in the
damaged DNA chain. T h e n an exonuclease
removes the damaged segment. A DNA polymerase next moves in to fill the gap, copying
from the opposite strand, just as it would in
ordinary replication. Finally, a DNA ligase
joins the new segment to the DNA chain to
complete the repair.
This sequence still essentially describes
the basic process, but it has turned out to be
much more complicated. It surely involves a
larger n u m b e r of steps. T h e multiplicity of
enzymes available in the cell's armamentarium
offers a seemingly endless array of repair
strategies.
In m a n y cases, for instance, it is now
clear that, in the removal of a thymine dimer,
what appears to be the first step is at least a
two-step process. As enzymologist Lawrence
Grossman of Johns H o p k i n s first showed,
before the endonuclease can cut a glycosylase,
it must first cleave the bond that connects
one of the thymines to its sugar. In E. coli
the incision step alone requires the cooperation of three genes. " T h e collaboration of
three genes in this m a n n e r , " S t a n f o r d ' s
I. Robert Lehman points out, "suggests that
DNA repair is under very strict genetic control indeed."
In h u m a n cells, the incision step is n o w
k n o w n to be controlled by at least seven
gene products, as is evidenced by the existence
of more than seven varieties of the XP defect.
(Cleaver, working with Dirk Bootsma and
his colleagues in Holland, was in on the discovery that by fusing the cells of two XP
patients, each of whose defects may involve
a different step or aspect of the incision
process, a hybrid cell is produced which
can carry out repair perfectly normally. T h e
deficiency in each is overcome by the strength
of the other.)
It turns out, too, that there are several
kinds of excision repair. Besides the excision
and replacement of damaged segments of
the DNA strand, there is also excision base
excision repair
Excision repair. To correct a thymine
dirner in the absence of light...
...endonuclease enzymes cut the dimer out of
the chain; exonucleases remove it...
...with the dlmer removed, polymerases
build a new segment out into the gap
from one end while ligases seal it into place at
the other...
...providing normal thymines to order
adenines into the complementary strand, thus
permitting replication to proceed.
SOS repair
Sloppy repair. If none of the usual mechanisms
can make the repair, and division is stalled...
...the sos repair system can be called into
being, sos polymerases are built from scratch
for the job. These enzymes jerry-build a
rough patch, containing random bases, which
permits the cell to survive and replicate.
But a mutation may well have been born.
repair, in which only the base is missing or
damaged, and excision nucleotide repair, in
which whole nucleotides—base, sugar, phosphate and all—are replaced, along with the
areas immediately adjacent. Base replacement is carried out by what are called insertases, a process discovered by Stuart Lynn
at Berkeley, while nucleotide repair is the
work of the same DNA polymerases Stanford
Nobelist Arthur Kornberg originally discovered. These are enzymes which, as Robin
Holliday of London's National Institute for
Medical Research has remarked, sometimes
act as if they had minds of their own.
Endless variations
Of the various kinds of response to DNA
damage, one of the most interesting is recombination, in which pieces of genetic material are traded from one DNA strand to
another. (Chromosomes invented recombinant DNA well before scientists did.) Not infrequently, gaps appear in a just-replicated
DNA strand—perhaps opposite damaged areas
that have been passed over. Fortunately there
are usually extra copies of DNA molecules,
or pieces of them, in the immediate vicinity.
Such an intact copy will move to overlap
with the damaged strand so that the necessary pieces can be spliced into the damaged
areas. The donor strand is then capable of
repairing its own damage so that, thanks
to the recombination process, all the DNA
molecules are now whole and healthy.
This recombinant capacity was not fully
realized until Paul H o w a r d - F l a n d e r s and
Dean Rupp of Yale noticed that cells that
were deficient in excision repair still managed to survive after undergoing a considerable amount of DNA damage. It was Evelyn
Witkin of Downstate Medical Center (now
at Rutgers) who demonstrated that the ultraviolet-induced mutations, that remain unrepaired in spite of everything, stem from
strand mismatching or copying errors made
while recombination is going on.
There are seemingly endless variations on
the theme of DNA repair, and scientists have
not yet begun to exhaust the catalog. If a
base is put in the wrong place during replication, there are enzymes to rectify the mistake. Purines, without any errors and without
damage inflicted from the outside at all, will
still drop out by the thousands every day
just from the ordinary wear and tear of
existence in the hectic molecular c o m m u n i ties we call chromosomes. W h e n they do
drop out, there may well be insertases to
replace them. There are no insertases, so far
as is k n o w n , to replace pyrimidines; they
drop out less readily.
What happens with reasonable frequency,
however, is that a base will spontaneously
undergo change; a cytosine, for instance,
will deaminate (lose an amino group) and
become uracil, another pyrimidine. Uracil
would be perfectly at home in RNA, the other
nucleic acid, but not in DNA. There do exist
enzymes (uracil glycosylases) that recognize
the uracil, remove it, and allow it to be replaced with a new cytosine. In case of a purine
(adenine or guanine) dropout, if it isn't re-
placed with reasonable s p e e d , the loosehanging sugar that no longer has its base
attached may start forming other compounds.
The result could conceivably be one of several kinds of cross-linking of the two strands
of DNA, which would involve another, more
difficult kind of repair.
Besides pyrimidine dimers, purine modifications (purines don't form dimers) can also
be formed—by the presence of mustard gas
or other chemicals, for example. In fact,
there are many kinds of damage caused by
X rays, gamma rays, cosmic rays and potent
chemicals. An a l k y l a t i n g agent such as a
nitrosamine can add an alkyl group—say, a
methyl or an ethyl group—where it doesn't
belong, and there are enzymes equipped to
remove those misplaced items.
O n the other hand, there are places on the
DNA chain where methyl g r o u p s do belong,
for good reason. S u p p o s e that an error has
occurred in one of the DNA strands—say, a
T has been put across from a G, where a
C really belongs. Well, there they are: two
strands, one with a G and one with a T. The
e n z y m a t i c a p p a r a t u s in the DNA-protein
complex " k n o w s " that c a n ' t be correct, but
how does it know whether to replace the C
with a T on one strand, or the G with an
A on the other? If the wrong strand were corrected, the result would be either the death
of the cell or a mutation, the perpetuation of
the genetic error. How can it be determined
which is the authentic original?
The answer was first suggested by Matthew Meselson of H a r v a r d . It appears that
MOSAIC January/February 1981
15
the older strand—the parent strand, the one
with the t r u e - b u t - m i s c o p i e d genetic m e s sage—processes strategically placed methyl
groups. But the newly formed strand, the
daughter strand, has not yet been methylated.
So the enzymes " k n o w " that the correction
is to be made in the new, unmethylated
strand.
T h e s e m e t h y l g r o u p s a p p e a r to serve
other purposes as well. Hamilton Smith of
Johns H o p k i n s and Werner Arber of the
University of Basel, Switzerland, who shared
a Nobel Prize in 1978, have s h o w n that bacteria can use methyl groups to distinguish
their o w n DNA from foreign DNA. If foreign
DNA intervenes, because it is unmethylated
or has methyl groups in inappropriate places
it is in danger of being chewed u p . Native
DNA nucleases—more of the c h r o m o s o m e s '
in-house, at~the-ready apparatus—will dismember it.
The chromosomes appear, then, to have a
protective recognition system of their own,
though how it works is still only dimly understood. There do exist recognition proteins,
enzymes called methylases, which may place
a methyl g r o u p on a DNA strand to protect
the strand from rampaging nucleases. As
Arthur Kornberg has observed, " Y o u can
get cells to commit suicide in tissue culture
by methyl-starving them." T h e DNA, in that
case, is attacked by its o w n nucleases, just
as the cells of a mature organism may be
attacked by its o w n lymphocytes in autoimmune disease.
The SOS system
Under certain circumstances, some cells
can switch on their SOS repair systems. T h e
term SOS denotes literally a holler for help,
for a rescue operation in an emergency. Much
about the SOS system is speculative. It appears
to be able to keep DNA synthesis going even
u n d e r d a m a g i n g attack b y r a d i a t i o n or
chemicals and in the absence of suitable defense enzymes. T h e n the cell seems to have
the p o w e r to call u p SOS polymerases—
enzymes which, in this case, it may synthesize ad hoc for this specific purpose. T h e
SOS enzymes slap a patch or a splice over
the damage so the cell will survive.
Of course a lot of mistakes are made this
way; the SOS system is also called the errorprone system or just " s l o p p y repair." But it
does save the life of a cell that would otherwise die. It's as if a victim of a serious
accident' is rescued, but winds u p with, say,
a limp, a couple of severed fingers, a hacking cough and the loss or damage of some
organ. T h o u g h his life has been preserved,
he isn't functioning very well and, handi-
16
MOSAIC January/February 1981
capped or crippled as he is, his life may
still be in jeopardy.
But he is still alive, and if he h a d n ' t been
rescued, he'd be dead. So it is with the SOSrepaired cell. It's a survivor with a number
of mutations that may or may not ultimately
prove to be deadly. Of course some kinds of
damage left over from the error-prone repair
system may still be repairable, post-replication, via the recombination process—just as
the man saved from death may well undergo
some post-rescue rehabilitation to improve
his condition.
And more
Still another repair p h e n o m e n o n is one
called host-cell reactivation. It is actually
just excision repair, b u t it is carried out on
a " g u e s t . " T h e cell repairs a DNA-damaged
virus introduced into the cell by an experimenter; the cell's genetic machinery repairs
the d a m a g e d viral DNA. In other c i r c u m stances, the guest can r e t u r n the favor.
Bruce Alberts's g r o u p at the University of
California in San Francisco is among those
seeking to elucidate this relationship.
Some cells are especially ultraviolet-sensitive—that is, repair-deficient—as first discovered by the late R u t h Hill at Columbia.
Such repair-deficient cells have been used
to good advantage by Bruce Ames of the
University of California at Berkeley to test a
variety of environmental mutagens and carcinogens. Other cells are particularly damage
resistant, and genes from a damage-resistant
cell can even impart their repair capacities to
a cell otherwise defenseless. Stephen Lloyd
of Philip Hanawalt's g r o u p at Stanford has
now, via recombinant-DNA techniques, been
able to clone a repair gene from a virus that
infects—and sometimes repairs—ultravioletdamaged DNA in £. coll. Lloyd hopes to
insert them into h u m a n XP cells that are
repair deficient. T h u s the XP cells may be
" c u r e d , " at least in tissue culture. Similar
experiments are being carried out at other
laboratories in Japan and the United States.
There need not be a specific repair technique to go with each type of DNA damage.
Some repair systems can fix more than one
kind of damage, and some kinds of damage
can be repaired by a n u m b e r of systems. As
for repair enzymes, they seem to keep proliferating with virtually every issue of the
relevant technical publications. The enzymologists keep proliferating too, despite the
fact that enzymes are devilishly difficult to
work with.
Johns Hopkins's Lawrence Grossman, for
instance, who with his collaborators has spent
a good many man-years purifying repair
enzymes, works with another bacterial model,
Micrococcus luteus. This bacterium is highly
resistant to DNA damage and possesses a
very low level of nonspecific nucleases—
hence very little b a c k g r o u n d noise—to interfere with the studies. Grossman has also isolated and purified a number of enzymes from
h u m a n placentas.
W h a t Grossman wants to do is to make
antibodies to each of his purified enzymes
to use as efficient probes. Radioactively labeled and dispatched into a system, each
antibody will seek out the enzyme to which
it is tailored and tell Grossman if any enzymes
are absent, are different, or are functioning
improperly, and where damage may be located
geographically in the nuclear material. In
this manner he hopes to be able to map
repair-enzyme functions with a precision
heretofore impossible.
Another veteran worker is Michael Lieberman (veterans can still be quite young in
this field) of W a s h i n g t o n University in St.
Louis, who elucidated the role of chromatin
in DNA repair in mammalian cells. And there
are the groups at Stanford—Hanawalt's, for
instance, and teams under Errol Friedberg
who is particulary interested in the glycosylases, and I. Robert Lehman who has made
a specialty of the DNA ligases. O n e could
go on enumerating them, not only in the
United States b u t in countries stretching
from the United Kingdom and Scandinavia
to Israel and the Far East.
Firefighters or fire wardens
Scientists—even those working actively
in the still infant science of DNA r e p a i r even as they get a better idea each day of
h o w much they still have to learn, are just
beginning to realize the enormous potential
of their explorations. As Hanawalt points
out, it takes a few thousand nucleotides to
make u p a h u m a n gene, and a single DNA
molecule may contain many thousands of
genes. All this m u s t be replicated every time
a cell divides, and at a fairly rapid rate.
The genes of £. coli replicate at the rate
of 3,000 nucleotides per second. T h i n k of
the fertilized egg of almost any multicellular
organism; and think of the number of times
it divides and reproduces its genetic information. T h i n k of the number of times, even
in a mature organism, that the genes have to
be switched on a n d off in order to carry out
the cell's basic processes—particularly protein manufacture.
And remember, Kornberg cautions, the
DNA molecule is not the relatively straightforward and relatively static structure it was
once thought to be. It is constantly coiling
and supercoiling, changing its shape and
configuration, moving dynamically in relation to its associated proteins, undergoing a
"virtual orgy of recombination," with plasmids arriving and departing like New Yorkt o - W a s h i n g t o n shuttles.
It is k n o w n n o w that genes " j u m p " and
"cross over," that pieces of chromosomes
are exchanged, that genes contain "silent"
sequences of DNA a m o n g the sequences that
are used, that DNA is being generated from
RNA by means of the enzyme reverse transcriptase, that segments of the DNA molecule
b u n c h u p in peculiar ways, raveling and
unraveling. (See " G e n e Segments on the
M o v e , " in this issue; "Split Genes: More
Questions Than Answers," Mosaic, Volume
10, N u m b e r 5; " T h e Structures That M a k e
DNA W o r k , " Mosaic, Volume 11, Number 1.)
Friedberg and Cleaver believe that some
failures of DNA repair may be due not to
the absence or malfunction of enzymes, but
rather to the d y n a m i s m of the DNA-protein,
or chromatin, complex and its failure to
open up at the appropriate times, thus perhaps
making the requisite gene sites inaccessible.
In any case, it is clear that in this frenetic
i n t r a n u c l e a r s i t u a t i o n , o p p o r t u n i t i e s for
damage and error are legion. And it becomes clear to Kornberg, the SOS system
notwithstanding, that DNA repair is perhaps
a misnomer. He proposes that it is not just a
h a n d y system to call on when in trouble, but
is rather a built-in part of DNA replication:
It is an unresting surveillance-and-editing
system that begins to work at the very beginning of life and does not stop until death,
though there is some evidence suggesting
that it declines with age.
Even the tiny mycoplasma, he observes,
an organism that hovers somewhere between
virus and bacterium, an organism with very
few genes, still reserves one of those genes
for repair. It's been calculated that a bacterial gene still has a 50 percent chance of
survival after having been duplicated 100
million times.
As Hanawalt, Lehman, Grossman, Setlow,
and others point out, life evolved on the
earth while exposed to a lot of ultraviolet
as well as other kinds of natural radiation
and a variety of damaging substances. So
repair s y s t e m s m u s t h a v e evolved along
with the earliest forms of life and probably
had an evolutionary influence of their own.
Errol Friedberg believes, in fact, that the
very reason for having DNA come in a double
helix was to make repair possible; for mere
replication, a single strand is quite sufficient,
though not as easy.
Does DNA actually repair itself? N o , says
A unisexfish
In the course of his research in excision
and other types of dark repair, Richard B.
Setlow performed an elegant experiment
in collaboration with Brookhaven National
Laboratory colleague Avery D. Woodhead
and Ronald W . Hart of Ohio State University.
O n e obstacle in the way of studying
DNA repair with the desired precision is
that most cells possess more than one
repair capability, so it is usually difficult
to tell what damage is being repaired by
which method. In his search for a better
way to study photoreactivation, especially
as it might apply to cancer, Setlow ran
across a remarkable experimental animal,
a small fish called the Amazon molly
(Poecilia formosa). It is so named not
because it comes from the Amazon region
(it is collected in northern Mexico and
southern Texas), but because it always
seems to come in the female gender. But
it does not reproduce parthenogenetically—
asexually; it requires the presence of male
sperm, which it can pick up from the water.
T h e presence of males of the common
tropical fishtank variety of black molly
serves the purpose adequately. The sperm,
though it is needed to activate fertilization, does not contribute any genetic material to the union. Thus only the female's
hereditary instructions are passed on to
the offspring. So mother, daughter, granddaughter, siblings, and all the other relatives through many generations are "isogenic"; they are, genetically speaking,
identical twins, a multigenerational clone.
T h e y do n o t , for i n s t a n c e , reject one
another's transplanted tissues. To render
P. formosa an even more desirable model,
the fish turn out to possess large quantities of p h o t o r e a c t i v a t i o n e n z y m e b u t
very little of any other kind of DNArepair capability.
Here, then, is the experiment Setlow
did with Hart and W o o d h e a d : They took
thyroid cells from the Amazon molly and
irradiated them with carefully measured
amounts of ultraviolet light. Then they
injected the damaged thyroid cells into a
number of healthy mollies. Virtually every
one of the recipient fish developed thyroid tumors.
T h e experiment was then done over;
only this time the damaged cells were exposed to visible light so that their photo-
r e a c t i v a t i o n e n z y m e s y s t e m s could be
activated before the cells were injected
into the recipients. This time only 5 percent of the fish developed tumors. Finally
the procedure was reversed, letting the
photoreactivation enzymes go to work
before the ultraviolet irradiation. This had
no protective effect; again the tumor incidence went back up to 100 percent. One
could hardly ask for a more clear-cut
demonstration.
This does not necessarily prove anything about h u m a n cancers, of c o u r s e even about h u m a n skin cancers. But it
surely suggests an important link of some
kind. N o one believes that ultraviolet
radiation damage, or DNA damage of any
kind, in itself causes cancer, but it makes
sense that such damage sets in motion a
series of events that can be carcinogenic.
Needless to add, Setlow is cultivating his
A m a z o n mollies for further experiments,
some already in progress. •
MOSAIC January/February 1981
1
A r t h u r Kornberg. It is the nuclear proteins,
especially the enzymes, that actively carry
out the repair on the passive DNA molecule.
DNA represents the library of information
that needs to be maintained with the greatest
possible accuracy.
Historians of contemporary genetics like
to smile at the old notion that the directive substance in the genes was not the nucleic
acid but the protein. Yet here are proteins
not only carrying out DNA repair, but " d e ciding" when to turn genes on and off at the
proper times to perform their daily functions. Is protein the directive substance, after
all, that controls the DNA? If DNA is the
library, is protein the librarian?
Kornberg believes the question is academic.
" W e need b o t h , " he says. "Protein can't
exist or replace itself without DNA's instructions. So it's a question like, ' W h i c h came
first, the chicken or the egg?' " The lusty
new science of DNA repair, then, not only is
making important contributions to knowledge of DNA in particular and genetics in
general, but it also offers new insights into
life's fundamental processes and raises profound philosophical questions.
Disease and aging
Nevertheless, perhaps of greater interest
to the average citizen is the great potential
for medicine. Xeroderma pigmentosum is not
the only genetic disease in which a DNArepair deficiency is implicated. There are at
least two other such diseases—ataxia telangiectasia and Fanconi's anemia—both with
varied symptoms but both producing a greatly
increased susceptibility to cancer. Here the
connection is not as clear cut as in XP, nor
is the specific repair deficiency the same as
inXP.
Repair deficiencies are also suspected in a
number of other genetic diseases—among
them D o w n ' s syndrome, hereditary retinoblastoma, Bloom's syndrome, H u n t i n g t o n ' s
disease and two diseases that mimic premature aging processes: Cockayne's syndrome
and progeria. Aging itself is believed in some
quarters to be associated with DNA repair.
There is a proposition that holds the decline
in DNA-repair capacity to be the major cause
of aging. As astute a student of the subject
as jerry Williams of George W a s h i n g t o n
University believes that the contention is
not incompatible- with the k n o w n data.
Evidence certainly points to the fact that
DNA repair does decline with age, but it is
more generally believed to be a s y m p t o m
rather than a cause of aging. Setlow and
Ronald Hart of Ohio State ran, among their
other experiments, a series on the cells of a
18
MOSAIC January/February 1981
n u m b e r of species of v a r y i n g life s p a n s
(shrew, mouse, rat, hamster, cow, elephant,
h u m a n ) and concluded that in general the
longer-lived species had greater DNA-repair
capacities.
Certainly DNA damage and repair are important in cancer. And repair studies will
surely constitute an indispensable tool for
all environmental medicine. DNA repair, as
it b e c o m e s better u n d e r s t o o d , may finally
tell, for instance, whether or not there really
is a safe threshold for radiation damage. It
m a y offer clues, too, to h i t h e r t o - u n s u s pected causes of birth defects. Walderico M .
Generoso and his colleagues at Oak Ridge
have s h o w n that, when a mouse egg is fertilized with a s p e r m a t o z o o n the DNA of
which has been deliberately damaged, DNA
repair begins right there in the egg.
A major hope, certainly, is that as understanding of DNA-repair mechanisms grows
more sophisticated, scientists may acquire
the ability to impart repair capability to cells
and organisms that are repair-deficient—or
even to those that aren't.
Such an ability could be important in addressing birth defects, cancer and possibly
even aging. Jerry Williams has already suggested that DNA damage, caused for instance
by free radicals, might be reversed or minimized by substances such as superoxide dismutase or antioxidants. And Joan SmithS o n n e b o r n of the University of W y o m i n g
recentlv did a series of experiments in which
she found, not surprisingly, that ultravioleti n d u c e d d a m a g e reduces the life s p a n of
paramecia. But when she followed the ultraviolet t r e a t m e n t s with p h o t o r e a c t i v a t i o n ,
and did so time after time after time, she
significantly increased the longevity of those
paramecia compared to that of unirradiated
controls. The only logical explanation seemed
to be that, while she was repairing the ultraviolet-induced damage, she was at the same
time repairing some of the ravages of aging.
While science seeks to make DNA-repair
systems more efficient, Michael Lieberman
w a r n s , the efficiency should stop s h o r t p e r h a p s far short—of 100 percent. T h o u g h
repair systems do need a high level of proficiency, he suggests, the imperfections in
the system may well be necessary too. For if
DNA-repair capacities were to become flawless, he reasons, then mutations would never
occur. If that were the case, h u m a n evolution would be brought to a screeching halt. •
The National Science Foundation contributes
to the support of the research discussed in
this article through its Genetic Biology, Cell
Biology, and Biochemistry
Programs,