dissection of functional domains in escherichia coli dna photolyase by linker-insertion mutagenesis

MolGenGenet (1992) 232:1-6

© Springer-Verlag 1992

Dissection of functional domains in Escherichia coli DNA photolyase by linker-insertion mutagenesis Kazuo Yamamoto

Biological Institute, Faculty of Science, Tohoku University, Sendal 980, Japan

Received March 29, 1991

Summary. The phr gene, which encodes protein of 472 amino acid residues, is required for light-dependent photoreactivation and enhances light-independent excision repair of ultraviolet light (UV)-induced DNA damage. In this study, dodecamer HindIII linker insertions were introduced into the cloned phr gene and the functional effects of the resulting mutations on photoreactivation and light-independent dark repair in vivo were studied. Among 22 mutants obtained, 7 showed no photoreactivation as well as no enhancement of light- independent repair. Four of these were located in amino acid residues between Gln333 and Leu371 near the 3' end of the gene, two were located in a small region at Glu275 to Glu280 near the middle of the gene and the remaining one was between Pro49 and Arg50. Three mutants that had insertions located in the 42 bp segment from 399 to 441 bp of the phr coding sequence (corresponding to amino acid residues Ile134 to Lys149) lost the light-independent repair effect but retained photoreactivation. These results suggest that (i) Escherichia coli DNA photolyase contains several critical sites that are distributed over much of the enzyme molecule, and (ii) a functional domain required for the effect on light- independent repair is at least in part distinct from that necessary for light-dependent photoreactivation.

Key words:phr gene - Dodecamer linker insertion mutagenesis - DNA photolyase - cis-syn cyclobutyl primidine dimer

Introduction

In Escherichia coli, damage produced by ultraviolet light (UV) is repaired by photoreactivation. This repair is mediated by DNA photolyase, a protein that acts specifi- cally on the major UV photoproduct, eis-syn cyclobutyl pyrimidine dimers (dimers). The enzyme consists of an apoenzyme (Mr=49000) and two chromophores, 1,5- dihydroflavin adenine dinucleotide (FADHz) and (5,10- methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). The enzyme binds to dimer-containing DNA indepen- dently of light and upon absorbing a 300-500 nm photon

breaks the cyclobutane ring of the dimer and thus re- stores dipyrimidines in the DNA (Sancar 1990).

If light is unavailable, photolyase-dimer complexes form but monomerization does not occur (Sancar and Sancar 1988). These complexes have been shown to have effects on DNA metabolism. DNA photolyase bound to dimers enhances the efficiency of excision repair by UvrABC (dark repair enhancement) (Yamamoto et al. 1983; A. Sancar et al. 1984), increases UV killing in excision defective cells (uvr potentiation) (Akasaka and Ya- mamoto 1991), blocks mutagenesis targeted at a dimer (Ruiz-Rubio et al. 1988; Yamamoto and Bockrath 1989) and enhances UV inactivation of lacZ gene expression (Li et al. 1991).

Comparison of the deduced primary structure of the photolyases derived from E. coli, Saccharomyces cerevisiae, Anacystis nidulans, Streptomyces griseus and Halo- bacterium halobium has identified one region near the C-terminus that is highly conserved and another near the N-terminus that contains a leucine zipper motif (G.B. Sancar et al. 1984; Yasui et al. 1988; Takao et al. 1989; Patterson and Chu 1989). These observations have led to speculation that such homologous regions are involved in chromophore binding and substrate modifica- tion.

Despite the importance of the enzyme for UV damage repair, only small amounts can be obtained from large quantities of normal E. coli cells. This has hampered detailed study of the structural aspects and functional domains of the protein. Very little is known about the interaction of the enzyme with its substrates or other DNA repair enzymes such as UvrABC nuclease. Also, little is known about the interaction of the peptide with its chromophores, FADH2 and 5,10-MTHF. Informa- tion of this nature would not only be of fundamental interest regarding the enzyme but would also be useful in understanding the mechanisms of action of dimer- binding proteins and flavoproteins. Since the DNA photolyase gene of E. coli, phr, has been successfully cloned (Sancar and Rupert 1978; Yamamoto et al. 1983), it is now possible to explore systematically the functional domains of this enzyme and investigate the significance of the evolutionarily conserved amino acid sequences by rapid and selective in vitro mutagenesis of the cloned gene.

We constructed a series of 22 HindIII linker-insertion mutations within the coding region of the phr gene and determined the effect of each mutation on photoreactivation, dark repair enhancement and uvr potentiation in vivo. The results indicate that mutants affecting photoreactivation map in three distinct clusters. We also obtained mutants that show photoreactivation but not dark repair enhancement and uvr potentiation. This suggests the existence of a functional domain that is needed for dark repair enhancement and uvr potentiation but not for photoreactivation.

Materials and methods

Bacterial strains and plasmids. The following strains are derivatives of E. coli K12:AB1157 (thr-i, ara-14, leuB6 A(gpt-proA)62, lacY1, tsx-33, supE44, galK2, his-4, rpsL31, xyl-1, mtl-1, argE3, thi-1) (Howard-Flanders et al. 1966) , KY1056 (ABI157 also recA56 srlC3OO:.'TnlO) (Yamamoto etal. 1984), KY1225 (AB1157 also recA56 srIC3OO: :TnlO phr-36) (Akasaka and Yamamoto 1991) and KY1226 (ABlI57 also uvrA6 malE.': TnlO phr-36) (Akasaka and Yamamoto). KY1056, KY1225 and KY1226 were constructed by P1 phage transduction as described (Ihara et al. 1985). Plas- mid pKY7102 is a derivative of pUC9 (Vieira and Mess- ing 1982) that contains about 1.6 kb of chromosomal DNA including the 1.4 kb coding region for E. coli photolyase (Fig. 1). All plasmids were grown in KY1225 and isolated by alkaline lysis (Birnboim and Doly 1979).

Media. L broth, L plates and K medium were used for growth of bacteria and were prepared as previously described (Yamamoto et al. 1985). Ampicillin was added at a concentration of 50 tlg/ml.

Chemicals. Restriction endonucleases, T4 DNA ligase and E. coli DNA polymerase (Klenow) were from Tak- ara Shuzo, Kyoto, Japan. Phosphorylated HindIII octamer linker was purchased from New England Biolabs, Mass., USA and dodecamer from Takara Shuzo, Japan.

Construction of HindlII linker-insertion mutations in the phr gene. The strategy used to construct a series of in- frame, linker-insertion mutations was based on the method of Stone et al. (1984). pKY7102 was partially digested with AluI, AccII, HaeIII, PvuII or RsaI to the extent that 20% to 50% of the circular DNA was con- verted to full-length linear DNA. The DNA was extract- ed with phenol, precipitated with ethanol, washed, resus- pended, and dodecameric HindIII linkers d(pCCCAAGCTTGGG), were ligated onto the blunt ends generated by each enzyme. After washing, drying, and resuspension of the precipitate, the DNA was digested with HindIII. The linearized full-length molecules (4.2 kb) were purified by preparative gel electrophoresis in low melting temperature agarose and ligated. A sam- ple of the DNA was used to transform competent KY1225 cells and ampicillin-resistant colonies were screened for the presence of plasmids containing a new HindIII site. Insertion of the octameric HindIII linker

d(pCAAGCTTG) was performed as described above, except that pKY7102 was partially digested with MluI or Sau3AI. In these cases, the 4 base sticky ends (5'CGCG3' for MluI and 5'GATC3' for Sau3AI) were first filled in with Klenow polymerase and these were ligated to the octamer linker. As a result, the plasmids acquired 12 bp at the MluI or Sau3AI site. The positions of HindIII linker insertion were then deduced by restriction mapping relative to the PvuII, MluI and/or BamHI sites and the known sequence of the phr gene (G.B. San- car et al. 1984).

Light-dependent repair and light-independent repair effects of the mutated phr gene. KY1225(phr-36 recA56) and KY1226(phr-36 uvr6) were transformed with plas- raids containing a HindIII linker insertion in the phr structural gene. UV irradiation and photoreactivation were performed as described previously (Akasaka and Yamamoto 1991). All manipulations besides the photoreactivating treatment were carried out under yellow light.

Immunoblot analysis. KY1225 cells harboring linker-insertion mutations were grown in 5 ml L broth at 37 ° C. Cells were centrifuged, suspended in 100 gl of DNA photolyase lysis buffer (50 mM TRIS-HC1, pH 8, 100 mM NaC1, 1 mM EDTA, 10 mM 2-mercaptoethanol) and sonicated. Cell debris was removed by centrifugation to obtain a crude cell extract. A portion of the extract was used to measure the total protein content by the Coo- massie Brilliant Blue protein assay kit (Bio-Rad, Calif., USA). The extracts were adjusted to contain the same amounts of total protein and were resolved by SDS 12.5% polyacrylamide gel electrophoresis (SDS-PAGE). Polypeptides in the gel were electrophoretically trans- ferred to a nitrocellulose filter, and probed with antibody to E. coli DNA photolyase in the presence of blocking solution. Detection was with alkaline phosphatase-con- jugated anti-rabbit IgG as the second antibody (Promega Biotec, Wis., USA).

Results

In frame insertion mutations

Mutagenesis was conducted on plasmid pKY7102 which contains the 1419 bp phr open reading frame and 168 bp of the upstream region. Structural features of the phr gene and the sites of linker insertions are shown in Fig. 1. The enzymes AccII, AluI, HaeII, RsaI, PvuII and Sau3AI were each used to cut pKY7102 in separate preparations to provide different distributions of linker insertions throughout the phr gene. Twenty-four of the linkers were within the phr gene, but the PvuII site overlapped with one of the AluI sites, and the MluI site was overlapped by one of the AccII sites. Thus, 22 different linker insertion mutations were obtained. We were able to isolate all of the potential insertions at the AceII, PvuII and RsaI sites but isolated only two of three at HaeII sites and five of six at Sau3AI sites. The nucleotide sequence of the phr gene was used to determine the amino acid

Funct ions Gene

UP DR PR

+ + + (Hae -35 )~ + + + (Acc-33)

+ + ÷ MIu 8 7

- - Acc 146 - -

+ + + Acc 276 - - + + + ACC 335 - -

+ Sau 399 - - + Acc 414 ~ ' - + Rsa 441

+ + + Sau 536 - -

-I- + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

Hae 717 - -

Alu 824 ---_.._J Ace 835 I

Sau 907 7 J Rsa 918 I Alu 9 9 8 - Sau 1071--___

pro te in --H/Pv

I

I I00

i

200

300

i .ae ' 5/I Sau 1 2 2 8 / - - | I Ace 1270 l l- I

Rsa 1 3 7 S - - I I / ~ : 1 Acc 1403--Lr ld<~ - J472

Fig. l. A schematic pKY7102, representing 1419 bp of the phr gene (open double line) and 168 bp of the 5' flanking region ofphr (filled line) in plasmid pUC9 (single line) HindIII-HincII sites, and the location of different linker insertion sites within the phr gene (numbers indicate base pairs from number 4-1, the first A of the ATG start codon). The corresponding amino acid residue is determined similarly (numbers are given taking the first Met as + 1). [This numbering is different from the original numbering of G.B. Sancar et al. (1984) in which the second Thr is taken as +1.] The presence (+) or absence ( - ) of photoreactivating effect (PR), dark repair enhancement (DR) and uvr potentiation (UP) in vivo is also indicated. Restriction sites: E, EeoRI and B, BamHI both came from pUC9 polylinker cloning sites; M/Hc, hybrid MluI- HincII junction, corresponding nucleotide number 1422; H/Pv, hybrid HindIII-PvuII junction, corresponding nucleotide number -168. Acc-33 and Hae-35 are mutants with a HindIII linker in the 5' upstream region of the phr gene and sometimes used as positive controls

sequence of each mutant protein that resulted from linker insertion.

Effects ofphr linker insertion mutations on photoreactivation, dark repair enhancement and uvr potentiation

Figure 2 shows examples of survival curves for strains carrying plasmids with different HindIII linker insertions in thephr structural gene, assayed before and after 5 min of photoreactivation. In Fig. 2a the host cell used has a defective phr allele, so alterations in survival rate asso- ciated with exposure to photoreactivating light indicates photoreaction mediated by the plasmid gene, e.g., strains with plasmid pKY7102 or mutant Sau399. In addition, dark repair enhancement is indicated by the degree of inactivation measured without photoreactivation. Mod- erate inactivation (with plasmid pKY7102) indicates enhancement and severe inactivation (with mutants Pvu1108 and Sau 399) indicates defective enhancement.

9_ 0 10 -1

'g. "g.

,~ 10-2

10-3 0

& .~ec A5,6phr 36 ,

1 2 3 Z,

b rec A56 phr ÷ i t I

1 2 3 UV ( J / m 2 ) ~ :

Fig. 2 a, b. Survival curves showing effects of different linker insertion plasmid mutants on photoreactivation and dark repair enhancement. After the indicated UV exposure, samples were exposed to continuous photoreactivating light for 0 min (open symbols) or 5 min (closed symbols) (day light fluorescent lamp filtered through polyvinyl plastic that cut out wavelengths below 380 nm). a KY1225(recA56 phr36) with: no plasmid (o, o); pKY7102(wild- type phr) (v, v); Pvull08 (zx, A); Sau399 (rn, i) . In this figure, only two representatives are shown. We also measured the photoreactivation ability and dark repair enhancement of the other 20 linker insertion mutants. The results are summarized in Fig. 1. b KYlO56(recA56 phr +) with: no plasmid (o, o); Pvull08 (zx, A); Sau 399 (D, n)

Seven mutants (Acc146, Alu824, Acc835, Alu998, Saul071, Acc1081 and Alul109=Pvu1108) lost both photoreactivation ability and dark repair enhancement. Mutant Acc146 has an insertion in the leucine repeat motif (Fig. 3). Mutants Alu824 and Acc835 are located near the possible dimer-binding site (Li and Sancar 1990) of the phr coding sequence (Fig. 4). Mutants Alu998, Saul071, Accl081 and Pvu1108 are located in the conserved region which shares homology with a variety of other photolyases (Takao et al. 1989) (Fig. 5). Three mutants (Sau399, Acc414 and Rsa441) retained full photoreactivation function but lost the capacity for dark repair enhancement. No significant characteristics have been reported about the sequences in these regions. The other 12 insertion mutants were as active in photoreactivation and dark repair enhancement as the wild-type phr plasmid.

The phr-proficient recA- strain (KY1056) was transformed with the plasmids carrying insertion mutations to see whether the mutant phenotype was dominant or recessive. As shown in Fig. 2b, plasmids Pvull08 and Sau399 had no effect on the UV sensitivity and photoreactivation ability of KY1056. Thus, the mutant proteins were recessive to wild-type DNA photolyase.

It is also known that amplified levels of DNA photolyase potentiate the UV killing'of excision repair-deficient cells in the dark (uvr potentiation) (Akasaka and Yamamoto 1991). As shown in Fig. 6, survival of different KY1226(uvrA6 phr-36) strains carrying plasmids

1 50 60 70 472

H. halobium M .... AHAAPPRVAFMLDALAALRERYRDLGSDLI .... E

A. nidulans M .... ADMAPARVAYLQGCLQELQQRYQQAGSRLL .... S

S. griseus M .... GFDAPNRLAFLADCLAALDAGLRHRGGRL I .... D

S. cerevisiae M .... HMDSGWKLMFIMGALKNLQQSLAELHIPLL .... M

E. coli M .... HNMSPRQAEL I NAQLNGLQ IALAEKGI PLL .... K + + + +

Acc146 Fig. 3. Comparison of the Escherichia coli Phr amino acid sequence with that of four photoreactivating enzymes at the proposed leucine repeat motif (indicates by + marks). Numbering is as described in Fig. 1. The Acc146 mutant that has PKLG inserted between P49 and R50 is indicated

i 270 280 472

H. halobium M---AAFIGQLAWREFYAQVLY---E

A. nidulans M- - -RVWQQELAWREFYQHALY- -- S

S~ g r i seus M- - -EAFVRQLAWRDFHHQVI A- - -D

S. cerevisiae M---QNFIKEVAWRDFYRHCMC---M

E. coli M---SVWLNELIWREFYRHLI T---K

Alu824 Acc835

Fig. 4 Comparison of the Escherichia eoli Phr amino acid sequence with that of four photoreactivating enzymes at the segment including the Trp278 (W278) residue, which has been suggested to be involved in dimer binding (Li and Sancar 1990). The positions of the Alu824 and Acc835 mutants are indicated. The residues that are conserved in the enzymes are marked by asterisks

o = 10-1

o

._> .> [ ]

10-2

V

1 0 - 3 I I t I

2.5 5.0 7.5 10.0 U V ( J / m 2 )

Fig. 6. Survival curves showing effect of different linker-insertion plasmid mutants on uvrA potentiating ability. Photoreactivation for 0 min (open symbols) and 5 min (closed symbols) was carried out as described in Fig. 2. KY1226 with: no plasmid (o, o); pKY7102 (v, v) ; Pvu1108 (A, A); Sau399 (D, I) . The results including the other 20 mutants are summarized in Fig. I

with representative HindIII linker insertion mutations indicated the distinctions that could be made. The seven plasmids that lost photoreactivation ability and dark repair enhancement when carried by the recA phr strain also lost uvr potentiating ability. The three mutants that lost dark repair enhancement but retained photoreactivation ability again had photoreactivation ability but did not potentiate the phenotype of the uvrA strain. Thus, deficiency or proficiency of the mutant plasmid for uvr potentiation paralleled that for dark repair enhancement.

Figure i summarizes the positions of the insertion mutants and the in vivo enzymatic activities of the mutants.

Synthesis of the mutant Phr proteins

Cell extracts were prepared from KY1225 harboring phr plasmids and investigated for the presence of proteins

corresponding to wild-type Phr in size by immunoblot- ting with anit-Phr antibody. The sizes of the mutant enzymes and the stability of the mutant enzymes against protease were not significantly different from the wild- type protein in all of the cell extracts (in Fig. 7, the results obtained from representative extracts are shown), suggesting that the mutant phenotypes were due mainly to altered conformation and/or function.

Discussion

Figure 1 and Table 1 summarize the different positions of the mutations produced by the insertion of four amino acids and the in vivo enzymatic activities resulting for each of the mutants. The activity of E. coil DNA photolyase is fairly tolerant of insertions. Of the 22 insertion mutants 12 retained full photoreactivating activity, dark repair enhancement and uvr potentiation in vivo, and 5 of these 12 have insertions within the carboxyl-

1 330 340 350 360 370 380 390 400 472 H. ha lob ium M---TGYPIVDAGMRQLRAEAYMHNRVRMIVAAFLTKDLLVDWRAGYD WFREKLADHDTANDNGGWQWAASTGTDAQPYFRVFNPMTQ---E A. n i d u l a n s M---TGYPIVDAAMRQLTETGWMHNRORMIVASFLTKDLI IDWRRGEQ FFMQHLVDGDLAANNGGWQWSASSGMDPKPL-RI FNPASQ---S S. g r i s e u s M- - -TGYPLVDAAMRQLAHEGWMHNRARMLAASFLTKTLYVDWREGAR HFLDLLVDGDVANNQLNWQWVAGTGTDTRPN-RVLNPV I Q- - -D S~. c e r e v i s i a e M---T•IPIVDAIMRKLLYTGYINNRSRM•TASFLS•MLLIDWRW•ERWFMKHLIDGDSSSNV••WGFCSST•DDAQPYFRVFNMDIQ---M E. eoli M---TGYPIVDAAMRQLNSTGWMHNRLRMI TASFLVKDLLIDWREGER YFMSQLIDGDLAANNGGWQWAASTGTDAAPYFRIFNPTTQ---K

** * *** ** ~'k ** ** * ** * * ~ * ~ * * ? * * * ~ * * * * * *

Alu998 Sau l07 i Accl081 P v u l l 0 8 Hael155 Fig. 5. Amino acid sequence homology between four photoreacti- tentiation, Alu998, Saul071, Acc1081 and Pvull08, are also indi- vating enzymes and that of Escherichia coli near the C-terminus. cated. For reference, the position of the mutant Hae1155 (between The residues that are conserved in all the enzymes are indicated A386 and A387) which is proficient for photoreactivating effect, by the asterisks. The four HindIII linker insertion mutants that dark repair enhancement and uvr potentiation is shown lost photoreactivating effect, dark repair enhancement and uvr po-

1 2 3 /-, 5 6 7 8 9

~!i~i ~ ii -q l

Fig. 7. Immunoblot analysis of phr HindIII linker-insertion mutants. Equal amounts (50 p,g) of crude extract from KY1225(re- cA56 phr36) transformed with wild-type and phr mutant plasmids were analyzed by using antiserum to an Escherichia eoli DNA photolyase. Lanes 1 to 7 contain extracts from KY1225(recA56phr-36) with plasmid Pvul 108, Acc835, Alu824, Sau399, Acc146, pKY7102 or pUC9. Lane 8 contains extract from the host strain KY1225. Lane 9 contains purified DNA photolyase (340 rig) as a 50 kDa marker, indicated by the arrowhead

proximal 20% of the protein (Hael155-Acc1403). This result suggests that four-amino acid insertions in the carboxyl-proximal 20% of the enzyme do not block three known functions of the protein.

The other 10 linker-insertion mutants, which map throughout the peptide, can be separated into two groups; one group lost photoreactivating function, dark repair enhancement and Uvr potentiation, and the other lost dark repair enhancement and uvr potentiation but retained the photoreactivating ability.

Depending on the reading frame and surrounding nu- cleotides (G.B. Sancar et al. 1984), different amino acids were inserted at the various sites (Table 1). All the insertion mutants reported here introduced similar changes in the hydrophobicity of the amino acids (data not shown). Therefore, the different functional phenotypes are most likely due to the Site rather than to the nature of the insertion.

To rule out the possibility of inadvertent small dele- tions or additions during linker insertion, which might produce frameshifts, or changes in the stability of the mutant protein, extracts of KY1225(phr) derived mutants were examined by Western blotting (Fig. 7). The sizes and amounts of the mutant proteins were found not to be measurably different from those of the wild- type protein.

The seven mutants that lost photoreactivation function, dark repair enhancement and uvr potentiation may be grouped into three classes: Acc146; Alu824 and Acc835; Alu998, Saul071, Acc1081 and Pvu1108. Payne et al. (1990) have demonstrated that the apoenzyme has no affinity for D N A but regained its specific ability to bind thymine dimer-containing D N A upon binding FAD. Therefore, these mutants may have defects in FADHz binding, D N A binding or both.

The Acc146 mutant has an insertion starting at base pair 146, which affects amino acid residues Pro49 and Arg50. As pointed out previously by Patterson and Chu (1989), this region contains features characteristic of the

Table 1. Peptide inserted into mutant proteins

Position of insertion Inserted amino acids

amino acid restriction site

R30-V31" Mlu 87 --A--S--L--A-- R30-V31" Acc 88 -P-S-L-S-- P49-R50 Acc 146 --P--K--L-G-- A93-E94 Acc 276 --Q-A-W-A-- A112-R113 Acc 335 --P-L-L-G- I134-L135 Sau 399 -Q-A-W-1- A139 V140 Acc 414 -Q-A-W--A- M147-K149 b Rsa 441 - (Y--~ S )--Q--A-W--D-- G179-S180 Sau 536 --S--K--L--S-- A240-$241 Hae 717 -Q-A-W-A- E275-L276 Alu 824 -P-K-L-G- R279-E280 Acc 835 -P-S-L-G- D303-R304 Sau 907 -P-S-L--D- V305-Q306 Rsa 913 -P-S-L-G- Q333-L334 Alu 998 --P-K-L-a-- I358-D359 Sau 1071 -Q-A-W-1- R361-E362 Ace 1081 -P-S-L-C~-- Q370-L371 Pvu 1108 --P--K--L--G- A386-A387 Hae 1155 -Q-A-W-A- D410-H411 Sau 1228 -P--S-L-D- R424-D425 Acc 1270 -P-S--L-G-- V459-Q460 Rsa 1375 -P-S-L-G- A468-R469 Acc 1403 -P--K-L-G-

Inserted amino acid sequences are given in the one-letter code

a The Mlu87 mutant inserts ASLA between R30 and V31. The Acc88 mutant inserts PSLG at the same position. Both the mutants are photoreactivation, dark repair enhancement and uvr potentiation proficient in vivo as shown in Fig. 1 b The HindIII linker at Rsa441 inserts QAWD between M147 and K149 and alters Y148 to S148

leucine zipper motif found in D N A binding proteins. The corresponding region in all other photoreactivating enzymes, including those of S. cerevisiae, A. nidulans, H. halobium and S. griseus, also contains the leucine repeats (Fig. 3). Insertions of the HindIII linker (resulting in insertion of Pro, Lys, Leu and Gly) may destroy the leucine repeat. It is therefore likely that the leucine repeats in this region are involved in D N A binding.

The Alu824 and Acc835 mutants (amino acid residues Glu275-Glu280) also lost photoreactivation, dark repair enhancement and uvr potentiation. Figure 4 shows the amino acid sequences of four photoreactivating enzymes at the region corresponding to the Alu824 and Acc835 insertion segment of the E. coli enzyme. Li and Sancar (1990) demonstrated that this region, corresponding to amino acid residues Trp278-Tyr282, was a likely binding region for damaged DNA. Thus, these two insertion mutants probably disrupt D N A binding resulting in defi- ciencies in photoreactivation, dark repair enhancement and uvr potentiation.

Four mutants, Alu998, Saul071, Acc1081 and Pvul 108 (corresponding to Gln333-Leu371), which have also lost photoreactivation, dark repair enhancement and uvr potentiation, were mapped at the highly conserved region of amino acid homology near the C-terminal (Fig. 5). Since we isolated the mutations by inserting four amino acids in this highly conserved region, the results confirmed the importance of the conserved region

near the C-terminal for D N A photolyase. As mentioned earlier, insertions within the carboxyl-proximal region (between Hael155 and Acc1403, corresponding to Ala386 to Arg469), where amino acid sequences are highly conserved, have no effects on photoreactivation, dark repair enhancement and uvr potentiation. In the near C-terminal conserved region, the carboxyl-proximal portion of the D N A photolyase is resistant to the insertion of four amino acids, but the carboxyl-distal portion of the enzyme is sensitive. We have at present no interpretation of this interesting result.

Three mutants, Sau399, Acc414 and Rsa441, corresponding to amino acid residues Ile134-Lys149, retained photoreactivation but lost dark repair enhancement. Thus, it seems there are two ways in which dark repair enhancement can be defective: when there is no or weak binding to substrate, or when a particular domain is altered, preventing a critical association with excision protein(s) even when bound to DNA. In order to determine whether these mutants have weak affinity for dimer and thus demonstrate photoreactivation during continuous light illumination but no dark repair enhancement, we did flash-photorepair experiments. These showed that strains carrying a mutant or wild-type plasmid re- sponded to flash-photorepair after UV radiation in es- sentially the same way (data not shown). Therefore, we assume that the mutant enzyme has a normal affinity for D N A substrate and chromophore but is defective for interaction with excision repair enzymes.

With regard to uvr potentiation, all the insertion mutants that lost dark repair enhancement lost uvr potentiation, and all the insertion mutants that retained dark repair enhancement retained uvr potentiation. The results suggest that the effects of bound photolyase on uvr potentiation as well as dark repair enhancement may require specific interaction(s) with some other molecule(s) in the cell rather than just a complex. Interaction between photolyase-dimer complexes and some other molecule in the cell has been suggested (Li et al. 1991).

Acknowledgements. We thank Dr. Richard Bockrath (Indiana Uni- versity) for several helpful comments during the preparation of the final typescript, and Dr. M. Ihara (Nara Medical University) for providing the purified E. coli DNA photolyase and rabbit antiserum against the enzyme. This work was supported by a Grant-in- Aid from the Ministry of Education, Science and Culture, Japan.

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Communicated by R. Devoret

dissection of functional domains in escherichia coli dna photolyase by linker-insertion mutagenesis

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