Life Sciences Vol . 16, pp . 1-6

Pergamon PressPrinted in the II .S .A .

MINIRBVIEiP

Pfi0T0RRACTIVATION IN ANIMAL CELLSBetsy M. Sutherland

Department of Molecular Biology and Hiochemietry

University of California

Irvine, California 92664

Ultraviolet light (220-300 nm) produces death and mutation in prokaryotes

and simple eukaryotee (1) and can induce skin cancer in man (2) .

The major

cause of ultraviolet light-induced damage in simple organisms--and hypothe-

sized cause of cancer induction--is the cyclobutyl pyrimidine dimer, formed

between adjacent pyrimidinea on the same DNA strand (1,3) . Cello have devel-

oiled three major pathways of circumventing the deleterious effects of dimers :

excision repair, recombination repair and photoreactivation. In eacieion

repair and recombination repair, multi-enzyme systems recognize and remove

dimers or other radiation-induced or chemically-induced lesions in DNA (1,4) .

In eacieion repair, dimers and other radiation or chemically-induced damage

are removed from the DNA by a combination of incision into one strand the

phosphodieater backbone, excision of the damaged region, and new synthesis

In recombinational repair,

bq a recombinational

(4) .

DNA repair processes

action of a single enzyme on a single substrate in a light-requiring reaction

(5,6) . The photoreactivating enzyme catalyzes the-monomerization of cyclobutyl

pyrimidine dimers induced in DNA by ultraviolet radiation (l1V) (7) .

As Figure 1 shows, the enzyme first binds to a dimer-containing region

of DNA . In the presence of visible or near ultraviolet light the enzyme

using the complementary strand as template (1) .

damage remaining after DNA synthesis is replaced

ism involving newly ayntheaized daughter strands

Photoreactivation, however, is unique among

breaks the cyclobutyl ring and returns the pyrimidines to their original

1

mechen-

in the

configuration . (See Figure 2)

Photoreactivation in Animal Cells

Vol . 16, No. 1

Su~a

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IV

0

FIG . 1

A schematic representation of W induction of damage to DNA And itsphotoenzymatic repair . DNA ie irradiated with ultraviolet light (220-300 net)producing pyrimidine dieters, shown here ae ~,

ltie photoreactivating enzyme(PRS) binds to the dieter-containing DNA, and in the presence of light in thewavelength range 300-500 net, monomerizes the dieter, thus repairing the DNA .

FIG . 2

A (diagramatic) comparison of the structure of DNA containing adjacentthymines with that of the thymine dieter in DNA . Dieters of thymine-cytosineand cytosine-cytosine pairs are also formed in DNA by ultraviolet light . thename cyclobutadipyrimidine has also been suggested for the dieter .

(8,9)

Photoreactivation is important as a DNA repair process, and ie of

physical-chemical intereet as an enzyme which requires light for catalysis .

In addition the specificity of the photoreactivating enzyme for pyrimidine

dieters (6) allows its use as an analytical tool :

if UV-induced biological

damage can be prevented by a true photoreactivatinn process, a major cause of

the damage was the cyclobutyl pyrimidine dieter . ltiis teat has been used to

show that in prokaryotes and in a simple eukaryote, Paramecium, dieters play a

Vol . 16, No . 1

Photoreaativatioa in Animal Celle

3

large part in the induction of death and mutation by W (1,10-12) . Although

it is important to evaluate the role of dieters in the induction of human skin

cancer by ultraviolet light, the photornactivation test had been unavailable

because the enzyme was thought absent from placental mammals (13) .

In fact, the species distribution of the enzyme eat quite striking : it

aaa present in all groups of all phyla except for the transforming bacteria

and placental masmalt . Cook (13) has rnvieaed extensively the data to 1971 on

the species and cellular distribution of the enzyme . Several possibilities

were suggested to account for this unusual distribution : the lots of the

enzyme in highly specialized or evolved groups, its absence in groups with

eaansiva sheltered embryonic development, or possible correlation of develop-

mental potential and loss of the enzyme . (See Ref . 13)

~e discovery of transformation in 8 . coli (14) (which is a photoreacti-

vable) removed the transforming bacteria from the exceptions to the rule of

universal photoreactivation .

Further, the finding of high levels of photo

reactivating enzyme in the orchid (Sutherland and Rnauft, unpublished results),

one of the most highly specialised plant families, indicated that epacialisa-

tion could occur without loss of photoreactivating enzyme activity . lheae

discoveries made the absence of photoreactivating enzyme in placental mammals

even more puzzling and pointed to an alternate hypothesis :

the enzyme might

be present but had not been detected because of technical difficulties .

Thin indeed turned out to be the case : a photoreactivating enzyme has

now been purified from human leukocytes (15) which meets all the criteria for

true enzymatic photoreactivation (16) . First, the enzyme requires dieter

containing DNA and photornactivating light for activity ; it causes the dis-

appearance of dieters from the DNA and converts dieter pyrimidinet to their

corresponding monomers . ~e activity is heat-labile and trypsin-sensitive

and is associated with a protein of molecular weight about 40,000 . ~e enzyme

has an ieoionic pH of 5 .4 and pH optimum of 7 .2 . All these properties are

similar to those of the yeast, algol and 8 . coli enzymes, those which have

4

Photoreactivation in Animal Cells

Vol. 16, No . 1

been studied in moat detail (17-19) . However, the human enzyme differs

strikingly in one requirement--the ionic strength optimum of the enzyme is

0 .05 (15), much lower than the 0.20 optimum of most other photoreactivating

enzymes (20) . If the human enzyme is asaeyed under conditions suitable for the

yeast or E . cola photoreactivating enzymes, the observed activity is only about

10-20% of that observed under optimal conditions . Since photoreactivation

assays are usually carried out at higher ionic strength, this peculiarity may

have accounted for previous negative results on photoreactivating enzyme in

placental mammals .

Photoreactivation in vivo

In many organisms, exposure to photoreactivating light after ultraviolet

irradiation greatly decreseee cell killing or mutation (1) . Aowever, data on

photoreactivation in mammalian cells are contradictory . Photoreactivation was

not found in the following cases : Chinese hamster cells tested for dieter

monomeriaation (21), UV-irradiated pseudorabiea virus grown on rabbit kidney

cells exposed to 90 min. of fluorescent light (22), and human and mouse cells

examined for photoreactivation of survival and DNA synthesis after a 10 min .

exposure to visible light (23) . On the other hand, adenine uptake by isolated

nuclei (24), and UV-induced damage to mouse ears and killing of the mouse (25)

were found to be photoreactivable . In addition, Pfefferkorn and Coady (22) and

Cook and Ryan (cited in 3) measured dieter monomeriaation in rabbit kidney and

human skin cells, respectively ; after long exposures to photoreactivating light,

about 15-20X of the dieters disappeared from the DNA . Since this activity was

much lees than in other cells tested at the same time (chick, potoroo and

wooly possum), these data were interpreted as negative .

Why should photoreactivation be so difficult to detect in intact cells?

First, in some cell types the enzyme may simply be absent . Second, in cells

with very efficient excision repair systems, photoreactivation mey be difficult

to detect over the large background of excision repair (26) . It has also been

suggested that the photoreactivating enzyme may have only limited access to

Vol. 16, No . 1

Photoreactivation in Animal Calls

the DNA of mammalian chromosomes (13) . Painter (personal communication) has

suggested that photoreactivatiog light may contain light detrimental to

mammalian cells which may mask beneficial effects of photoreactivation .

In addition to masking of actual photoreactivation, apparent photorecovery

effects may, in reality, not result from true enzymatic photoreactivation . In

photoreactivation, visible light administered before UV-irradiation gives

apparent photo-recovery effects (27) ; it is thought that a growth delay induced

by the photoprotecting light may allow more time for light-independent repair .

P'hotoreactivating light may also have effects on hormonal cycles in intact

animals, which may affect growth or viability thus making it very difficult to

discern the true effects of photoreactivation (13) .

Why is the detection of photoreactivation in mammalian cells important?

First, it is important to be able to assess the biological role of the photo-

reactivating enzyme and determine its contribution towards the repair of normal

cells . Second, the specific and exclusive action of the photoreactivatiog

enzyme on pyrimidine dimera allows its use ae an analytical tool : if the

biological damage caused by UV can be prevented by true enzymatic photoreacti-

vation, a major contributor to the damage was the dieter . The demonstration

that human cells do possess photoreactivatiog enzyme may thus allow a direct

aseeeement of the role of dieters in the induction of human cancer by ultra-

violet light .

Raferencee

1. R. B . SETLOW, Science 153 379-386 (1966) .

2. J . ft . EPSTEIN, _In Photophyeiolog9 (edit . by A. C . Giese), Vol V, p. 235-273, Academic Prass, New York .

3. R. HART and R. B . SETLOW, Abstracts , _Amer. Soc . for Photobiology , letAnn . Meeting (1973) .

4 . P. HOWARD-FLANDERS, Ann . Rev. Biochem. 37 175-200 (1968) .

5 . C . S . RUPSRT, J . Gen. Phyeiol. 43 573-595 (1960) .

6 . J . &. SETLOFI and R. B., Nature 197 560-562 (1963) .

7 . R . B. SETLOW, W. L. CARRIER and F . J. BOLLUM, Proc . _Nst . Açad . Sçi . _U .S .A .53 1111-1118 (1965) .

Photoreactivation in Animal Cells

Vol . 16, No . 1

8 . W . E . CORN, N . J . LEONARD and S . Y . WANG, Photochem , Photobiol . 19 89-94(1974) .

9 . J . J . MADDEN, H . WERBIN and J . PENSON, Photochem , Photobiol . 18 441-445(1973) .

10 . B, M . SUTHERLAND, W . L . CARRIER and R . B . 3ETLAW, Biophya . J . 8 490-499(1968) .

11 . B . M . 3UTHERIAI~, W . L . CARRIER and R . B, SETLOW, Scieace .. 158 1699-1700 .

12 . R . F . KIMBALL, Mut . Ras . 8 79-89 (1969) .

13 . J . S . COOK, In Photophysiology (edit . by A . C . Giese) V III 191-233Academic Press, New York .

14 . N-G AVADHANI, B . M . MEHTA and D . V . REGE, J . Mol . Biol . 42 413-423 (1969) .

15 . B . M . SUTHERLAND, Nature 248 109-112 (1974) .

16 . B . M . SIITHSRLADID, DNA Re ir, (P.C . Hanawalt and R . B . Setlow, eda,) inthe press, Plenum Press, New York (1975) .

17 . A, MUHAPAIED, J . Biol . Chem . 241 516-523 (1966) .

18 . N . SAITO and H . WERBIN, Biochemistry 9 2610-2620 (1970) .

19 . B . M . SUTHERLAND, J . C . SUTHERLAND and M, J . CHAMBSRLIN, J . Biol . Chem .284 4200-4205 (1973) .

20, J . S . CO~C, In Molecular and Cellular Repair Processes (R, F . Beera, R . M .Herriott, andR. C . Tilghmân, ede,) pp . 79-94 Johns Hopkins, Baltimore,

21 . J . E . TROSRO, E, H, CHU aad W . L . CARRIEß, Radiation Res . 24 667-672,

22 . E . R . PFEFPERRORN and H . M . COiADY, J . Virol . 2 474-479 (1968) .

23 . J . E, CLEAVER,~ Biochem . Bio

e, Res . Commun . 24 569-576 (1966) .

24 . R . LOGAN, M . ERRERA and A, FICA, Biochim , Biophys . Açta 32 147-155 (1959) .

25 . A . F . RISCR and S . CARLSON, J . Cell . Comp . Physiol . 46 301-305 (1955) .

26 . W . HARM, C . S . RUPERT and H . HARM, In Molecular and Cellular Re airProcesses (R . F . Beere, R . M . Herriott and ß . C .Tilg man, edâ~, 53-63) Johns Hopkina, Baltimore,

27 . J . JAGGEß, Introduction to ßeeearch in Ultraviolet Photobiology , Prentice-Hall, Englewood Cliffs, New Jersey (1967) .