Restriction enzyme cleavage of ultraviolet-damaged simian virus 40 and pBR322 DNA

J. Mol. Biol. (1983) 170, 305-317

Restriction Enzyme Cleavage of Ultraviolet-damaged Simian Virus 40 and pBR322 D N A JAMES E. CLEAVER

Laboratory of Radiobiology and Environmental Health University of California, San Francisco, CA 94143, U.S.A. (Received 19 January 1983) ('leavage of specific DNA sequences by the restriction enzymes EcoRI, HhldIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects wm~ attributed to cyclobutane pyrimidine dimem in the recognition sequences: the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. (!ombining EcoRI with dimerspecific endonuclease digestion revealed that pyrimidine dimem blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimem along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and (' bases. 1. Introduction Restriction enzymes depend for their function on precise recognition of specific palindromic sequences in DNA and precise cleavage at defined sites (Modrich, 1979). The recognition cleavage patterns can be altered by a variety of perturbations to DNA sequences, including the incorporation of base analogs (Berkner & Folk, 1977: P e t r u s k a & Horn, 1980: Modrich & Rubin, 1977), specific or random methylations (Boehm & Drahovsky, 1980: Modrich, 1979: W o o d b u r y etal., 1980), and ultraviolet radiation damage (Cleaver etal., 1982: Hall & Larcom, 1982). In this paper I describe further investigation of the dose-response relationships between photoproducts produced by u . v . t and restriction e n z y m e cleavage, and the relationship between the DNA strand containing the dimers and the EcoRI cleavages t h a t are permitted or blocked. 2. Materials and Methods Simian virus 40 (SV40) (strain 776) supercoiled DNA was prepared from monkey CV-I cells infected at high multiplicity and harvested after 72 h growth in 0-1/xCi [14C]dThd/ml t Abbreviations used: u . v . , ultraviolet light: dThd. thymidine. 3O5 0022-2836/83/300305-13 $03.00/0 .C" 1983 Academic Press Inc. (London) Ltd.

306

J. E. CLEAVER

(56 ('i/mol) by means of the Hirt ext,~ction method (Williams & Cleaver, 1978). Plasmid (pBR322) supercoiled DNA was prepared fi'om Escherichia coli (strain RRI) infected with plasmid pBR322. The SV40 DNA consisted of single-length molecules, and the pBR322 contained a major fraction (>90~)) of tandemty duplicated molecules. Both DNAs were purified by cesium chloride/ethidium bromide equilibrium density centrifugation (Williams & Cleaver, 1978) and the supereoiled DNA band was collected. Ethidium bromide was extracted with isopropanol, and the DNA was dialyzed to remove cesium chloride. The DNA was precipitated with 2vol. ethanol and then dissolved with Tris-Mg buffer (100 mMTris. HC1, 5 mM-MgC12, 2 mM-2-mercaptoethanol, 50 mM-NaC1, pHT.2) at approximately 300 t~g/ml (pBR322) or 100/~g/ml (SV40). Samples (10 tLl) of DNA were irradiated with u.v. (1"3 J m -2 s- l at 254 nm) at fluences h'om 0-8 x l02 to 31"2 × l()2 J m -2 immediately before digestion with various restriction enzymes (TaqI, BamI, SalI. EcoRI, HindIII and HhaI : Bethesda Research Laboratories). DNA samples (1 to 3 t*g in l0 gl) were incubated with the enzymes (10 to 100 units per t~l) for approximately 1 to 2 h at 37o( '. To ensure complete cleavage of all possible sites, the enzyme/DNA ratio was approximately 10-fold higher than the minimum needed for complete digestion of 1 tLg DNA. The enzyme reaction was quenched by adding 5 gl of E,ado P~ Stop solution (Na2EDTA. 5o~b (w/v) sodium dodecyl sulfate, 25% (w/v) glycerol, 0-025% (w/v) Bromophenol blue; Bethesda Research Laboratories). Some samples were incubated for 30 min at 37°C with 5 t~l of a crude u.v. endonuclease, prepared from Mic~vcocctls ltde~ts (Zelle et al., 1980). to cleave DNA at pyrimidine dime,' sites. For this reaction, the Tris-Mg buffer was adjusted by adding a final concentration of 10m~I-EDTA to block non-specific nuclease action. Samples were electrophoresed in horizontal 0-8°b (w/v) agarose gels tbr 6 h at 100 V in borate buffer (100mM-Tris, 3 mM-Na~EDTA, 90mM-boric acid. pH 8-3). Gels were stained in 0"5t~g ethidium bromide/ml and photographed. Radioactivity was determined in 2 to 5-mm slices by melting the agarose in glass scintillation vials before dissolving in water-miscible counting solution. Non-radioactive gels were quantified by microdensitometry of photographic negatives : there was an approximate linearity between densitometric reading and DNA content within the range of measurements used.

3. Results (a) Cleavage of u.v.-irradiated pBR322 DNA with T a q I

TaqI has seven distinct cleavage sites in pBR322 D N A : after irradiation, or digestion a t low e n z y m e / D N A ratios, n u m e r o u s f r a g m e n t s were produced a t sizes ranging downward from a p p r o x i m a t e l y twice full-length linear molecules (Fig. 1). Because the starting plasmid D N A was p r e d o m i n a n t l y a t a n d e m l y duplicated population, f r a g m e n t s larger t h a n single-length linear molecules were observed at very low enzyme concentrations or high u.v. fluences (Fig. 1). T h e ladder of f r a g m e n t s obtained after digesting u.v.-irradiated pBR322 D N A at high e n z y m e / D N A ratios was the same as t h a t obtained after digesting control pBR322 D N A at low e n z y m e / D N A ratios. Both the size classes and their relative intensities were similar, implying t h a t each of the seven cleavage sites was affected similarly b y u.v., t h a t u.v. d a m a g e merely blocked cleavage r a n d o m l y , and t h a t no new cleavage sites were introduced by D N A damage. However, whereas extending the time of incubation at low e n z y m e / D N A ratios increased the e x t e n t of cleavage in unirradiated DNA, the range of f r a g m e n t s seen a t high e n z y m e / D N A ratios in u.v.-irradiated D N A represented the limits of digestion, and longer times did not change the p a t t e r n s of sizes (data not shown).

RESTRICTION 1

2

CLEAVAGE 3

4

OF u.v.-DAMAGED 5

6

7

DNA

307

8

Fl(:. 1. Restriction fragments from control and u.v.-irradiated pBR322 (tandemly duplicated) DNA incubated with Taql. Left to right: cham~els 1 to 4. control DNA incubated with TaqI at decreasing enzyme/DNA ratios (20 units//~g, 10 units/#g, l unit/gg, and 0-1 unit/#g DNA, respectively) for 5 min at 37°C: channels 5 to 8, u.v.-irradiated DNA (fluences of 0.8 × 102, 3.9x 102, 11.7 x l02, and 31.2 x l02 J m-2. respectively) incubated with TaqI at a high enzyme/DXA ratio (20 units/gg).

308

J. E. ( ' L E A V E R 1

2

3

4

5

6

7

8

9

l"m. 2. Restriction fragments fi'om control and u.v.-irradiated (l-6xlO2,lm -~) SV40 I)NA incubated with DNase 1 or EcoRI. ('hannels 1 to 4. SV40: channel 1, control sul)er(..oiled f(wm I DNA: channel 2. control form II DNA prepared by incubation with DNAse I (2 gg/ml) in ethidium bromide (10()/~g/ml): chamael 3. control form III DNA l')rel)ared by incuhation with EcoRl (IOunits/tzg): channel 4. u.v.-irradiated DNA incubated with EcoRl (lOunits/p.g). ('hannels 5 to 9, pBR322 (tandemly duplicated): channel 5. control form 11 DNA prepared by incubation with l)NAse I (2 tzg/ml) in ethidium bromide (1()(1#g/ml): channel 6. control form I l I DNA prepav'ed hy incubation with EcoRl (10 units//~g): channel 7. u.v.-irradiated DNA incubated with EcoRl : channel ,~. (,ontrc~l form I1 DNA l)repamd by ineuhation with HindlII (1(1 units/tLg): channel 9. u.v.-irradiated I)NA incubated with Hindlll (1()units/#g). A similar study using a higher gel com.entration (1('.) was reported previously (('leaver el al.. 1982). (b) ('lea~,age of St'40 a~d pBR322 D N A with single-site enzymes When U.v.-irradiated SV40 (single-length) DNA was incubated with EcoRI (for w h i c h t h e r e is a s i n g l e site) t h e e x p e c t e d s i n g l e - l e n g t h l i n e a r ( f o r m I I I ) m o l e c u l e s w e r e p r o d u c e d , b u t t h e r e w e r e a l s o r e l a x e d c i r c u l a r ( f o r m I I ) m o l e c u l e s ( F i g . 2).

RESTRICTION

CLEAVAGE

OF u.v.-DAMAGED DNA

309

T h e s e m u s t arise from c l e a v a g e o n o n e s t r a n d o n l y , as p r e v i o u s l y d e m o n s t r a t e d (Cleaver et al., 1982). T a n d e m l y d u p l i c a t e d p B R 3 2 2 D N A i n c u b a t e d w i t h EcoRI or HindIII, for which t h e r e are two d i a m e t r i c a l l y o p p o s i t e c l e a v a g e sites in t h e t a n d e m molecule, g e n e r a t e d t w o m a i n classes of m o l e c u l e ( s i n g l e - l e n g t h a n d d o u b l e - l e n g t h linear) a t low to m o d e r a t e u.v. fluences (Fig. 2). T h e s e arise from c l e a v a g e a t b o t h H i n d I I I sites in t h e u n d a m a g e d m o l e c u l e s b u t a t o n l y one site in m o l e c u l e s c o n t a i n i n g u.v. d a m a g e . I n s i n g l e - l e n g t h molecules, therefore, u.v. d a m a g e p r e v e n t e d c o m p l e t e c l e a v a g e b u t did p e r m i t c l e a v a g e on one s t r a n d . I n t a n d e m l y d u p l i c a t e d p B R 3 2 2 , n e u t r a l gel e l e c t r o p h o r e s i s did n o t resolve w h e t h e r t h e r e c o g n i t i o n site t h a t w a s b l o c k e d fi'om c o m p l e t e c l e a v a g e c o n t a i n e d a b r e a k o n o n e s t r a n d , b u t t h i s is likely, c o n s i d e r i n g t h e results with s i n g l e - l e n g t h D N A . V a r i o u s r e s t r i c t i o n e n z y m e s i n c u b a t e d w i t h single or d o u b l e - l e n g t h m o l e c u l e s p r o d u c e d a v a r i e t y of i n c o m p l e t e c l e a v a g e p r o d u c t s ( T a b l e 1), c o n s i s t e n t w i t h t h e view t h a t u.v. d a m a g e b l o c k s c l e a v a g e w i t h i n r e c o g n i t i o n s e q u e n c e s in a t least o n e of t h e s y m m e t r i c a l l y l o c a t e d c l e a v a g e sites.

(c) Efficiency of cleavage by various restriction enzymes T h e p r o p o r t i o n o f D N A m o l e c u l e s in which c o m p l e t e c l e a v a g e w a s b l o c k e d w a s d e t e r m i n e d for s e v e r a l single-site r e s t r i c t i o n e n z y m e s a f t e r i r r a d i a t i o n o f p B R 3 2 2 ( t a n d e m l y d u p l i c a t e d ) D N A . A t t h e s a m e u.v. fluence, i.e. t h e s a m e a v e r a g e n u m b e r of photoproducts, cleavage by HindIII was blocked much more TABLE 1

Restriction enzyme ,specificity and classes of DNA remaining after enzymatic cleavage of irradiated DNA (single-length S V 40 or tandemly duplicated pBR322) that contains one or two cleavage sites Enzyme

BamI EcoRI HindIII SalI TaqI HhaI

Recognition sequence G-G-A-T-C-C C-C-T~A-G-G G-A-A-T-T-C C-T-T-A-A-G A-A-G-C-T-T(T) T-T-C-G-A-A G-T-C-G-A-C C-A-G-C-T-G C-T-T-C-G-A-T-T G-A-A-G-C-T-A-A G-C-G-C C-G-C-G

DNA

FI

Classes remaining after cleavaget FII FII (2 x ) FIII FIII (2 x )

SV40 pBR322 SV40 pBR322 pBR322

+~ +$

+ + -

+~ +$

+ + + + +

+ + +

pBR322

-

-

-

+

+

SV40

+

+

-

+

-

SV40

-

-

-

+§

-

+, Visible band: - , no band, Fluenees of u.v. up to 23"4 x 102 J m-Z : some weak bands are visible only at high fluences. # FI. form I. etc.: 2 x, tandemly duplicated double-length molecules. $ Faint bands from double-length pBR322 circles and higher multimers (see Fig. 4). § Of 2 linear fragments of 4736 and 490 base-pairs produced by HhaI, only the larger was observed, with no effect of u.v. up to 23'4 x l02 J m -z

J . E. ( ' L E A V E R

310

1

2

3

4

Fit;. 3. Restriction fragments fl'om control and u.v,-irradiated (1"6 x 102J m -2) pBR322 (tandemly duplicated) DNA incubated with HindIII or B a m I (3(X}units/t~g}. Channel 1, control DNA incubated with HD~dIII: channel 2. u,v.-irradiated DNA incubated with HindIII: channel 3, control DNA incubated with Banzai: channel 4, u.v.-irradiated DNA incubated with BcamI,

RESTRICTION CLEAVAGE OF u.v.-DAMAGED DNA

311

efficiently t h a n was cleavage by BamI (Fig. 3). This implies t h a t there m a y be different frequencies or kinds of p h o t o p r o d u c t s in the respective recognition sequences. HindIII recognizes a sequence containing a C-T-T. T h e r e is an additional T residue on one side of the hexanucleotide sequence (Sutcliffe, 1978) in pBR322, producing a short pyrimidine isostich t h a t would be a " h o t s p o t " for c y c l o b u t a n e pyrimidine d i m e r production. BamI recognizes a sequence containing T-C-C, which would have a far lower pyrimidine d i m e r yield t h a n the HindIII site, even though it is a potential site for production of (6-4)pyrimidine-cytosine, non-cyclobutane dimers {Lippke et al., 1981 ; Franklin et al., 1982). T h e difference in the e x t e n t of inhibition of HindIII and BamI cleavage is therefore an indication of the n u m b e r and kinds of p h o t o p r o d u c t s produced within the recognition sequences. F o u r enzymes, EcoRI, HindIII, TaqI and H h a I , were chosen for more extensive investigation. (d) Dose-response relationships between extent of cleavage

and pyrimidine dimers SV40 and pBR322 ( t a n d e m l y duplicated) D N A were irradiated with u.v. over a wide fluence range and t h e n incubated with enzymes t h a t recognized single sites in the respective single-length molecules from the set EcoRI, HindIII and TaqI. SV40 was also incubated with H h a I , which m a k e s two closely spaced cuts in SV40; only the larger 4736 base-pair piece was observed in the 0"8 to 1% agarose gels under the conditions used. The yield of pyrimidine dimers was also determined b y thin-layer c h r o m a t o g r a p h y of 3H-labeled pyrimidines from SV40 irradiated under the s a m e conditions as used before e n z y m e digestion. F o r both SV40 (EcoRI and TaqI) and pBR322 (HindIII) the fraction of molecules incompletely cleaved was a linear function of the pyrimidine d i m e r yield (Figs 4 to 6). Cleavage by HhaI was complete t h r o u g h o u t the fluence range. TABLE 2

Efficiency of u.v. in blocking restriction enzyme cleavage

Enzyme

DNA

No. of dimer sitest T-T T-C

EcoRI HindIII TaqI

SV40 pBR322 SV40

2 6 1

3 4 2§

Hhal

SV40

0

0

Blocked molecules: dimers$ 3"3_ 0"9 12-8+- 1.3 6"7_+0-7 (FI I) 0"88+_0"25 (FI) 0

t Number of dimer sites (T-T and T-C, or C-T) with at least one of the pyrimidines within the recognition sequence. This is estimated fi'om the total number of dimer sites in the recognition sequence multiplied by 2 for the tandemly duplicated pBR322, No correction has been made for the varying efficiencies with which dimers may be produced according to adjoining sequences (Haseltine et al., 1980). :~Slope of regression lines (Figs 5 and 6) +_standard error. FI, form I. etc. § If dimers in flanking sequences 3 base-pairs from the cleavage sites, without respect to the strand specificity, are included, the total number of dimer sites, T-T, T-C, and C-T, is 6.

o

~

E

2

m

~

3

-"

to

4

d)

¢M

5

4"

*,.-

6

-:

~

7

g.

m

8

_-"

~.

9

~_

~0

10

.

_¢'

~

11

~r

12

Fio. 4. Restriction fragments from pBR322 (tandemly duplicated) DNA incubated with HindIII (300 units/~g DNA) after increasing doses of u.v. Channel l, tandemly duplicated form II (FII) DNA prepared by incubation with DNAse I (2 #g/ml) in the presence of ethidium bromide (100 #g/ml), and other species, as denoted, resulting from partial digestion. Channel 2, unirradiated DNA incubated with HindIII. Channels 3 to 12, DNA irradiated with u.v. fluences as given ( x l02 J m-2).

F=(IX)

F]]Z

FI

FTI

"

z

1

RESTRICTION

CLEAVAGE

OF u . v . - D A M A G E D

DNA

313

8 I0

o

E

O

E

2 ~5

5

O

O

o

Q.

1.0 2.0 3.0 Percentoge of thymme-dimers (3H, T=T/T) FI~;. 5. Percentage uf I)XA in form 11 molecules after incubation of 3Hqabeled SV40 with EroR[

and Hlml after increasing fluem'es of u.v. (O) Ec,JRI : ( Q ) Hb,:tI.

The slope of the regression lines relating tile percentage of blocked molecules to the l)ercentage of dimers is a measure of the relative efficiency with which dimer yields correlated with the inhibition of restriction enzyme cleavage (Table 2). The slope of the lines can be regarded as a measure of the number of effective targets within the recognition sequences. These efficiencies were very close to the total nnml)er of T-T and T-(' cyclobutane pyrimidine dimer sites in the recognition regions for H i n d I I I in tandemly duplicated pBR322 and for EcoRI in singlelength SV40. HhaI recognizes G-('-G-(' sequences and was unaffected by very high fluences. TaqI generated both form II and form I molecules as approximately linear functions of fluence. The yield of form II molecules was greater than

0

8 I5 i:l

E 10 2 "5

u

1.0 2-0 3.0 Percentage of thymine-dimers (3H, /'=T/T) |~'l~, 6. Percentage o|" I)NA ill |'~rnl ] and |'ornl II m(>[ecu[es after [ncuhaticm o|' SH-labeted SV40 with Tltql after inc~x-asing fluenc,es of u.v. ( 0 ) F o r m II; ( 0 ) form I.

J . E. ( ' L E A V E R

314

05I 0.4

_A

0-5

2

0.2

o

"6

,~

0.I

Form 1T

E

400 J/m z

¢!

!

/

I >

Linear

xlO

2

5

4

5

e" 6

7

8

9

0.6

10 II •

o,5

It~~~7ooJ/mz II

0-4

o.3

I//

0.2

I

S, l

o., I

2

3

4

5

6

7

8

9

I0 /I

Gel slice number

l"l(;. 7. 3H radioactivity in gel slices of agarose gels obtained after incubation of 3H-labeled u,v.-irradiated ( 4 x l 0 z and 7 x l ( ) Z J m -z) SV40 DXA with EcoRI followed by M. httelm u,v. endonuclease. (O) EcoRI only: (O) EcoRI followed by endonu(.lease.

expected from the number of potential dimer sites, and the yield of form I molecules was less than expected (Table 2).

(e) Relative orientations of photoproducts and cleavage sites in SV40 Cleavage of u.v.-damaged SV40 DNA with EcoRI generated fully cleaved linear molecules and form II molecules containing a single nick. Because of the linear dose-response relationships (Figs 4 to 6), the form II molecules were most likely produced by a single photoproduct within the recognition sequence. This raises tile question of tile cis-trans relationship between tile photoproduct and the permitted and prevented nicks within the recognition sequence. In an attempt to answer this question, u.v.-damaged SV40 was cleaved first by EcoRI, and then by M. h~teus u.v. endonuclease, in the presence of EDTA to block non-specific nucleases. In control experiments, the dimer-specific activity was clearly demonstrated by extensive nicking of u.v.-damaged form I SV40 molecules (not shown), but EcoRI produced no change in the distribution of form II and

RESTRICTION

CLEAVAGE

Subsfrote

OF u.v.-DAMAGED

U.V. damaged

Control

Fill

CX::X:3
Fill O

FII

( b ) Single site

duplicated

Fill

( c ) Multiple

,~ites FI

A~B B

A~B B ,C

C

C" A • c

C

¢

Multiple A--B sites duplicated • ¢ A

(d )

cl l ! 2xFI

( + F i l and 2 x F I T ~ 2xFIII \ a t high doses /

............Fill

~::]Cx:x:~O3C~2XFI

~

315

Rest ricfion products

( o ) Single si'ie

A

DNA

A •

i

c

~ Additional

'" C A

B A J fronm~=n÷s

Arab

e

C A

C

C

A

A

B

C A

A B

C

A

II

g

C

B C

\

A

ac AE. . . AS ~ C

C

A

A n

Ii

C

A

A

A

C

~

C

C

A A

•

II

g

B

C

Additional

~ragments A

• B c

C A

A ~ •

Fi(:. ~. Interpretative scheme for possible products of complete or partial cleavage of control and u.v.-irradiated form I DNA (single or tandemly duplicated) by r~striction enzymes that cleave at one or multiple sites per DNA molecule. (a) Single form I molecules cleaved by enzymes that recognize single sites per molecule. (b) Tandemly duplicated form I moleculescleaved by enzymes that recognize single sites in unduplicated molecules. (c) Single tbrm I mo]ectfiescleaved by enzymes that recognize multiple sites per molecule. (d)Tandemly duplicated form I molecules cleaved by enzymes that recognize multiple sites. A. B and ('. cutting sites. Additional fl-agmeuts am the fragments observed as a consequence of partial cleavage as seen with TaqI (Fig. 1). form I molecules produced by EcoRI fl'om 3H-labeled SV40 (Fig. 7). I f the dimer and the single EcoRI cleavage in u.v.-damaged SV40 had been on opposite strands, then cleavage by u.v.-specific enzyme activity would have converted the form I I molecules to lineal' molecules. Because this was not observed, it seems reasonable to conclude t h a t the dimer and the single EcoRI cleavage were on the same strand, and t h a t the dimer blocked cleavage on the opposite strand.

4. Discussion This s t u d y indicates t h a t the introduction of u.v. d a m a g e into D N A has extremely local and well-defined effects on restriction enzyme function. The results permit estimation of the range of influence of p h o t o p r o d u c t s both within the D N A strand containing them and on the opposite strand. Not only are these results useful for understanding restriction enzyme function, b u t they m a y

316

J. E. CLEAVER

provide insights into protein-DNA interactions and the way damaged sites disturb these interactions to produce wide varieties of altered cellular function. Consideration of the possible alternative molecular species produced by inhibition of single or multiple cleavage sites by u.v. can provide a satisfactol\v framework for interpreting these results (Fig. 8). The efficiency of u.v. in blocking cleavage by EcoRI and Hi~dIII appears to be a direct consequence of the number of potential dimer sites within recognition sequences (Table 2). Each recognition sequence contains several potential dimer sites that constitute multiple independent targets for u.v. damage (Figs 4 to 6 and Table 2). The HindIII recognition region has an additional flanking thymine, producing a T-T-T sequence which is a hot-spot for dimer production (Haseltine et al., 1980: Lippke etal., 1981). The actual dimer yield will therefore be slightly above the average for the entire DNA. This would account for the high efficiency of production of blocked, uncleared molecules relative to the average dimer yields. The EcoRI site contains only two T-T sites, the others being T-C or C-T, which form photoproducts less efficiently by u.v. than T-T. The actual dimer yield will therefore be slightly below the average for the entire DNA. Damage by u.v. appeared to block cleavage by EcoRI on one strand but not on the other, producing form II molecules. The combination of EcoRI with M. l~le~s u.v,-specific endonuclease activity indicated that the blocked cleavage sites were all trans to the dimers for this restriction enzyme (Fig. 7). Pyrimidine dimers consequently blocked cleavage within one base-pair on the opposite strand, but failed to influence the cleavage site three or four bases away on the same strand. Therefore, without reference to strand specificity, it seems that dimers have a range of influence of at least one base-pair but less than three. TaqI cleavage of u.v.-irradiated pBR322 DNA (Fig. 1) produced a large variety of partially cleaved molecules, as expected fl~om the number of different possible fragment sizes that can be produced by partial cleavage of tandemly duplicated molecules (Fig. 8). Cleavage of u.v.-irradiated SV40 DNA, which contains a single TaqI site, produced both singly nicked form II and unnicked form I molecules as linear functions of fluence (Fig. 6). Both the qualitative and quantitative results with TaqI were different from those obtained with EcoRI and HindIII. However, if the principle developed fi'om EcoRI and HindIII experiments is applied to the results from TaqI experiments, the efficiency of u.v. in blocking cleavage (Table 2) should be an indication of the number of effbctive sites for u.v. damage in the sequence that constitutes the target for TaqI action. Form I molecules will be observed because lesions completely blocked cleavage, and the observed efficiency of 0.88-1-0.25 implies that one photoproduct is sufficient. The TaqI recognition sequence is a tetranucleotide, and a T-C photoproduct site would straddle the cleavage site on one strand and be within one base-pair of the cleavage site on the other strand. Therefore. it seems reasonable to conclude that cleavage by TaqI is blocked at both sites by a single photoproduct at either of the T-(' sites. The production of form II molecules by TaqI implies that photoproducts at other sites than those implicated in form I production are sufficiently close to block cleavage only at one of the cleavage sites. From the base sequence around the TaqI site of SV40, six dimers can be formed within three base-pairs of the

RESTRICTION

C L E A V A G E OF u . v . - D A M A G E D

DNA

317

cleavage sites (Fiers etal., 1978). Because these dimers are not in pyrimidine isostichs, they are not produced in a b o v e - a v e r a g e a m o u n t s by u.v. Therefore, to explain the results obtained with TaqI on the basis of flanking pyrimidine dimers produced by u.v., it is necessary to consider t h a t TaqI is sensitive to distortions in D N A caused by p h o t o p r o d u c t s up to a b o u t three base-pairs a w a y from the tetranucleotide recognition sequence. TaqI, therefore, m a y recognize its tetranucleotide cleavage sequence with a specificity t h a t depends on the integrity of a larger sequence of DNA. An a l t e r n a t i v e consideration for the TaqI o b s e r v a t i o n s is the recent recognition t h a t there are u.v. p h o t o p r o d u c t s in D N A other t h a n the c y c l o b u t a n e dimers. These involve T-C sequences, which are in the TaqI recognition sequence (Wang, 1976; W a n g & Varghese, 1967: Lippke et al., 1981), a n d are formed in some sites with efficiencies larger t h a n expected on the basis of average p h o t o p r o d u c t yields. In addition, they can have greater biological effects t h a n pyrimidine dimers (Lippke et al., 1981; Haseltine et al., 1980; Brash & Haseltine, 1982; LeClerc & Istock, 1982). The effects of u.v. on cleavage of the TaqI site and other sites containing T-C sequences therefore merit f u r t h e r investigation as a w a y of elucidating possible roles of n o n - c y c l o b u t a n e photoproducts. This work was supported by the U.S. Department of Energy. R E FEREN('ES

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