THE JOURNAL OF BIOtOGICAL CHEMISTRY Vol. 261, No. 12, Issue of April 25, pp. 5663-5673,1986 Printed in U.S.A.

Cloning of the Bacteriophage T4 uvsX Gene and Purification and Characterization of the T4 uvsX Recombination Protein*

(Received for publication, October 4, 1985)

Deborah M. Hinton and Nancy G . Nossal From the Section on Nucleic Acid Biochemistry, Laboratory of Biochemical Pharmacology, National Znstitute of Arthritis, Diabetes, and Digestiue and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

The bacteriophage T4 UVSX gene is a nonessential gene required for normal levels of DNA repair, recom- bination, and replication. We demonstrate that plas- mids containing the T4 DNA approximately 300-2900 base pairs upstream of T4 gene 41 express a biologi- cally active uvsX protein. This UVSX protein imparts increased survival to UV-irradiated T4 UVSX- phage and decreases the T4 uusX- mutant suppression of a conditionally lethal T4 mutant in the gene 49 recom- bination nuclease.

The uvsX protein purified from cells with a UVSX’ plasmid catalyzes ATP hydrolysis to ADP and AMP and, in the presence of the T4 gene 32 helix-destabliz- ing protein, ATP-dependent strand exchange between homologous circular single-stranded and linear duplex DNA, These results agree with the recent characteri- zation of uvsX protein from T4-infected cells by Yone- saki et aZ. (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H. (1985) Eur. J. Biochem. 148,127-134) and by Formosa and Alberts (Formosa, T., and Alberts, B. M. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 363-370). Tn addition, we find that under some reaction conditions strand exchange is catalyzed by UVSK protein in the absence of 32 protein.

The level of the uvsX protein expressed by the UVSX’ plasmids is high and independent of the orientation of the T4 DNA within the vector. This suggests that tran- scription promoter($ lie upstream of the UVSX gene on the cloned T4 DNA. In vitro transcription of T4 DNA restriction fragments reveals two tandem promoters whose transcripts initiate approximately 500 and 600 nucleotides upstream of the UVSX gene and extend through the gene.

The genome of bacteriophage T4 is a linear duplex of 166 kilobase pairs which is circularly permuted and terminally redundant (1, 2). Genetic analyses of T4 mutants displaying altered DNA synthesis have implicated many phage genes in DNA replication and metabolism (3, 4). Generally, these mutants have been classified as belonging to one of the following three categories: DNA- (no DNA synthesis), DNA delay (delay in onset of DNA synthesis), and DNA arrest (early stop of DNA synthesis). In vitro studies (5-7) have demonstrated that the products of six DNA” genes (gene 43- DNA polymerase, 44-, 62-, 45-polymerase accessory proteins, 32-helix-destabilizing protein, and 41-primae component)

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

plus one DNA delay gene (61-primase component) together catalyze efficient strand displacement synthesis of DNA on a nicked circular template and prime discontinuous synthesis on the displaced strand. These studies suggest that these seven T4 gene products are the proteins required in vivo for new DNA synthesis on the leading and lagging strands of a replication fork. As DNA synthesis proceeds in uiuo, the main form of T4 DNA observed is a complex network (8) which sediments faster than T4 DNA extracted from the phage (9). Mosig and eo-workers have proposed (reviewed in Ref. 10) that this network arises from strand invasion by the single- stranded DNA present at the unreplicated terminus of one DNA molecule into the homologous duplex region of another T4 genome. It is proposed that the invading single strand is then extended by the complex of replication enzymes. Since the T4 genome is circularly permuted, such a recombination/ replication process would result in the type of network of highly branched concatenated structures observed for repli- cating T4 DNA.

The T4 genes needed for the observation of the T4 concat- enated structures constitute a recombination pathway (dis- cussed in Refs. 11 and 12) which is thought to include (among other T4 genes) uusX, uusY, uvsW, and gene 49. Proposals concerning the functions of these gene products are based in part on the following evidence. First, in vivo, conditionally lethal T4 gene 49 mutants, under nonpermissive conditions, accumulate more complex networks of T4 DNA (13) with even higher sedimentation values (14, 15). Extracts from T4 49+ infections, but not from T4 49-, convert this DNA to material which sediments more slowly (14). Furthermore, the purified gene 49 protein, a DNA endonuclease (16, l?), cleaves DNA substrates containing cruciforms or Holiday structures at the branch point to yield products which can be sealed with DNA Iigase (18). Taken together, these studies support the proposal that the gene 49 protein is required to resolve the recombinational network into structures which can be proc- essed for packaging (13, 14, 18). The nonessential T4 genes, uusX, UUSY, and uus W, are needed for the wild type level of resistance to UV and ionizing radiation and chemical agents, implicating these gene products in DNA repair (for a review, see Ref. 19). In addition, these genes are also needed for normal T4 recombination. Mutants in each of these three genes display decreased recombination frequencies (11, 20, 21), and uusX or uusY mutants have a DNA arrest phenotype (22, 23) consistent with their inability to continue normal DNA synthesis which is thought to proceed through recom- bination intermediates. Mutations in uusX or uvsY also sup- press gene 49 mutations (22-24), and a UUSX- (or UUSY-) 49- double mutant infection does not produce the very fast sedi- menting DNA observed after 49- infections (25). These results

5663

5664 Cloning of T4 uvsX Gene

support the hypothesis that the uvsX and uvsY gene products contribute to the formation of the concatenated structures which require 49 nuclease for processing (12,25). Presumably, a UUSX- 49- or uvsy - 49- mutant survives by packaging the few T4 progeny genomes produced by replication without the recombination intermediates.

A more precise role for the uvsX gene product in recombi- nation has been elucidated by the biochemical studies of Yonesaki et al. (26) and by Formosa and Alberts (Ref. 27, cited in Refs. 28 and 29). These workers have purified the uvsX protein from T4-infected cells and report that this protein is a single-stranded DNA-dependent ATPase that promotes ATP-dependent homologous pairing and strand ex- change of DNA. These DNA-pairing reactions are analogous to those carried out by the Escherichia coli recA protein. Griffith and Formosa (29) have recently shown that, again like the recA protein, uvsX protein binds cooperatively and tightly to double-stranded or single-stranded DNA to form filaments which can be observed by electron microscopy. Taken together, these i n vitro observations suggest that the UVSX protein, through its promotion of homologous pairing, contributes directly to the formation of the concatenated structures of replicating T4 DNA observed in vivo.

Genetic mapping experiments have assigned the UUSX gene upstream of gene 41 (22,30). We have located gene 41 on the physical map of T4 by showing that plasmids containing T4 map units 22.01 to 20.06 express 41 protein (31). In the process of constructing plasmids containing gene 41, we have found that the region upstream of gene 41 encodes a protein of about 46 kDa (this paper). We show that this protein is the T4 uvsX gene product and that the presence of a plasmid expressing uvsX protein in the cell complements T4 UUSX- phage. In addition, we have purified the protein expressed by a uusX+ plasmid to homogeneity. Our preliminary characterization of this protein indicates that, like uvsX protein from T4-infected cells, it is a ssDNAl-dependent ATPase and a dsDNA-binding protein which promotes strand exchange between linear du- plex and homologous single-stranded DNA.

* EXPERIMENTAL PROCEDURES~

RESULTS

The T4 DNA Upstream of Gene 41 Expresses a Protein with an Apparent Molecular Weight of 46,000-We have previously shown (31) that the plasmid pDH428 (T4 DNA from map units 24.3 to 20.06 (Fig. 1, A and B) contains gene 41 in the region between the BglII site at 22.01 and the Hind111 site at 20.06. Although 41 protein is expressed by pDH428/DHl cells at a level that can be easily detected in a complementation assay €or DNA synthesis (31), the level of the protein is not high enough to observe a 59-kDa band co-migrating with 41 protein on a Coomassie Blue-stained polyacrylamide-SDS gel (Fig. 2, lanes 7 and 15). However, comparison of the proteins expressed by cells with pDH428 with those expressed by cells

The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; bp, base pair(s); T4 dC-DNA, cyto- sine-containing T4 DNA SDS, sodium dodecyl sulfate; kDa, kilo- dalton(s); RF, replicative form.

“Experimental Procedures” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-3319, cite the authors, and include a check or money order for $4.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

containing the vector (lane 1) does reveal the presence of a band of another size (46 kDa) that is apparently dependent on the T4 DNA sequence. To determine whether this new protein is encoded by the T4 DNA, we constructed several plasmids (Fig. lA) which include part of the 4500 bp of T4 DNA cloned into pDH428. We then asked whether cells containing these plasmids also express the new protein. As shown in Fig. 2 (lane 141, cells with the plasmid pDH428A1, in which 3980 bp of T4 and vector DNA downstream of the BglII site have been deleted, produce the 46-kDa band. This result suggests that only the T4 DNA region upstream of the BglII site is necessary for expression of this protein. Further- more, production of the protein, at a level high enough to be observed in these crude extracts, must require at least part of the 360 bp of T4 DNA between the XbaI site (map unit 22.37) and the BglII site (map unit 22.01) since deletion of this region (pDH428A2) eliminates the protein band (lane 12).

Direct evidence that the protein is encoded by the T4 DNA upstream of gene 41 was obtained by observing the in vitro translation products expressed by plasmids containing this region or expressed by T4 DNA restriction fragments (Fig. 3, A and B) . Supercoiled plasmid DNA or linear T4 DNA restriction fragments were transcribed and translated in vitro in the presence of [35S]methionine using an E. coli 8-30 extract. Transcription/translation of the vector plasmid (panel A, lane 2) gives one product of 34 kDa. We assign this protein as p-lactamase, the product of the amp‘ gene which is present in the pBR322 sequence and has been previously shown to be a major in vitro translation product of pBR322 (46). Insertion of the T4 DNA within the vector to yield the recombinant plasmids pDH428 (panel A, lanes 3 and 4) and pDH447 (panel A, lane 5 ) results in plasmids whose major translation product is a highly expressed species of 46 kDa. The 46-kDa band is again observed as the major species if just the T4 DNA from this region, a Sal1 restriction fragment (map units 24.8 to 20.75) or an EcoRI fragment (map units 24.3 to 21.15) is transcribed and translated in vitro (Fig. 3B, lanes 2 and 3). These results support the previous in vivo results indicating that the gene for the 46-kDa protein is present on the T4 DNA.

We have inferred the direction of the 46-kDa gene based on the translation products expressed by the plasmid pDH428A3, a plasmid identical to pDH428 except that the 800 bp between the ClaI sites a t map units 22.85 and 22.00 have been deleted (Fig. hi). The 46-kDa protein is not made by this plasmid in vivo (Fig. 2, lane 9) or in vitro (Fig. 3A, lane 6) . However, i n vitro, the major species is a highly expressed new protein of 30 kDa. We interpret the 30-kDa band as an abnormal protein generated by the fusion of the open reading frame of the 46-kDa protein to new DNA se- quences. In addition, we assume that this fusion has occurred by joining the DNA encoding the N terminus of the normal 46-kDa protein to new sequences, rather than fusing the DNA of the C terminus to another start. This assumption is rea- sonable since the 30- and the 46-kDa proteins are similarly expressed at a very high level and represent the major trans- lation products of pDH428A3 and pDH428 (or pDH447), respectively. Since the plasmid pDH428Al produces normal levels of the 46-kDa protein i n vivo (Fig. 2, lane 14), the gene for this protein cannot begin downstream of the BglII site at map unit 22.01. Thus, we conclude that the production of the abnormal 30-kDa protein by pDH428A3 indicates that the normal gene starts upstream of the CZaI site at map unit 22.85 and proceeds toward gene 41. This direction is consistent with the previously determined general direction of early T4 tran- scription in vivo (2), the direction of the immediately down-

Cloning of T4 uvsX Gene 5665

EcoRI

pDH428

Bglll

NsiI

Hind111

FIG. 1. The bacteriophage T4 genome in the region of genes 41,61, and uvsX. Panel A shows the T4 DNA from a SalI site at map unit 24.8 through gene 61. (Map units refer to those in Ref. 58 and represent approximately 1000 bp.) The positions of genes 41 and 61 have been determined previously (31, 32,59, 60). The identification and approximate position of the uusX gene is given in this paper. The position of the UVSX gene is denoted by dashes to emphasize remaining uncertainties in the exact position of the 5' and 3' termini of the gene (see "Fksults"). The position of two E. coli RNA polymerase promoters active in uitro are designated as k and have been located upstream of uvsX (see Fig. 4). Beneath the restriction map, the regions of T4 DNA inserted in various plasmids are shown by open bars. Solid bars denote deleted regions. Details of the plasmid constructions are given under "Experimental Procedures" and in Ref. 31. (pDH428A1 represents a deletion of DNA in pDH428 between the BglII site at 22.01 and the NdeI site within the vector shown in panel B. Thus, the vector DNA present in this plasmid differs from the other plasmids shown.) Restriction sites referred to in the text are shown and have been determined using T4 dC-DNA and/or the plasmids containing cloned T4 DNA from this region. We have observed differences from the reported restriction sites (58) in the region between the EcoRI site at map unit 24.3 and the XbaI site at 22.37. We do not observe a Pstl site reported to lie at map unit 22.40 after restriction of the purified T4 SalI fragment (map units 24.8 to 20.75) or restriction of the plasmids. In addition, our calculation of distances between the EcoRI site at 24.3 and the XbaI site at 22.37, again using the purified SalI fragment and the plasmids, differs from reported values (58). We have indicated our values of these distances which were used to determine the start of the in vitro transcripts (see text). Panel B shows the plasmid pDH428 (31) which contains T4 DNA (I) from the EcoRI site at map unit 24.3 to the HindIII site at map unit 20.06. The T4 DNA has been ligated to a fragment of the gal expression vector pKG1810 (37) which contains E. coli sequence (0) with the IS2 transcription terminator (T) and the galK gene followed by pBR322 sequence (-) from 2068 to 4363 (61). amp' designates the ampicillin resistance gene expressing p-lactamase.

stream genes 41 and 61 (31,32), and the position of the major upstream of the ClaI site a t map unit 22.85 (enough sequence promoters in this region as determined in vitro (see below). t o encode a protein of 30,000 daltons). Since the 46-kDa Based on the size of the abnormal pDH428A3 product (30 protein is observed in vivo in cells containingpDH428Al (Fig. kDa), we estimate that the farthest upstream position for the 2, lane 14) but is not observed in those containing pDH428A2 5' end of the 46-kDa gene (assuming no introns) is 820 bp (Fig. 2, lane 12), we conclude that the 3' end of the gene

5666 Cloning of T4 UVSX Gene

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

\ \ \ I 1 1 1 1 1 \ \ I .“=”rS I / / - ,

68- - - c c r r

- 41 protein t. -” . - E

- 68

C- 46 kDa protein -? I b

43 - - 43

L==GGsa

c c

FIG. 2. Cells containing plasmids with the T4 DNA up- s t r eam of gene 41 express a 46-kDa protein. Proteins of crude extracts obtained as described (32) were separated on 10% polyacryl- amide-SDS gels. The positions of the 46-kDa protein, of gene 41 protein, and of markers having molecular weights of 68,000 (bovine serum albumin) and 43,000 (ovalbumin) are indicated. Crude extracts were from DH1 cells containing the following plasmids: lune I , pKG1810; lane 2, pLS502; lane 3, pLS501; lane 4 , pDH4426; lune 5 , pDH447; lane 6, pDH4112; lane 7, pDH428; lane 8, pDH421; lune 9, pDH428A3; lane IO, pDH428A5; lune 11, pDH428A4; lune 12, pDH428A2; lane 13, pDH447; lune 14, pDH428Al; and lune 15, pDH428. See Fig. 1A for DNA contained in each plasmid.

probably lies between the XbaI site at map unit 22.37 and the BglII site at 22.01. Thus, we position the gene for the 46-kDa protein upstream of gene 41 on the T4 genome as indicated in Fig. lA.

In Vitro Transcription Demonstrates That the T4 DNA Upstream of the 46-kDa Gene Contains Two Promoters for E. coli RNA Polymerase-In the previous translation experi- ments, we observed a high level of the 46-kDa protein both in vivo and in vitro. In addition, the expression of the protein is independent of the orientation of the T4 DNA within the plasmid (Fig. 2: compare pDH4426 with pDH447 (lanes 4 and 5 ) and pDH428 with pDH421 (lanes 7 and 8)). These results suggest either that the T4 DNA contains its own transcription promoter(s) for the expression of the 46-kDa gene or (less likely) that the gene is translated a t a very high level from message generated by read-through transcription from the vector. To test whether the T4 DNA we have cloned contains promoters active in vitro, we transcribed the T4 SalI fragment (map units 24.8 to 20.75) and the EcoRI fragment (map units 24.3 to 21.15) using E. coli RNA polymerase in the presence of [m-”P]UTP. In order to favor specific transcription starts from stronger promoters, we transcribed the DNA using a low molar ratio of RNA polymerase to the DNA template and we challenged the RNA polymerase-DNA binding by the addition of heparin 2 min before the addition of the triphosphates (see “Experimental Procedures”). Use of either the SalI or the EcoRI fragment yields large RNA run-off transcription prod- ucts shown on the 1% agarose gel in Fig. 4A. However, because this gel is nondenaturing the sizes for the RNAs cannot be determined, although the major species from transcription of the EcoRI fragment appears to be shorter than that obtained after transcription of the SalI template. To determine the size

68

4 1 protein

46-kDa protein

43’

30

A

1 2 3 4 5 6

B 1 2 3

68 -

41 protein-

48-kDa protein \““

43 ”

30-

- FIG. 3. T h e T4 DNA upstream of gene 41 expresses a 46-

k D a protein in vitro. Plasmid DNA or linear T4 DNA restriction fragments were transcribed and translated in vitro in the presence of [35S]methionine as described under “Experimental Procedures.” Panel A shows a 10% polyacrylamide-SDS gel of the translation products obtained after translation using: lune I , no DNA; lane 2, 1000 ng of pKG1810R3; lune 3, 330 ng of pDH428; lune 4 , 50 ng of pDH428; lune 5 , 650 ng of pDH447; or lane 6, 330 ng of pDH428A3. Punel B shows the products after translation using: lane I , 50 ng of pDH428; lane 2,50 ng of T4 4330-bp Sal1 fragment (map units 24.8 to 20.75); or lane 3,40 ng of T4 3400-bp EcoRI fragment (map units 24.3 to 21.15). The positions of the 46-kDa protein as well as those of I4C-labeled standards (molecular weights of 68,000 (bovine serum albumin), 43,000 (ovalbumin), and 30,000 (carbonic anhydrase)) and gene 41 protein are indicated.

of the major transcript(s), we cleaved the SalI fragment with the restriction endonucleases BglII, XbaI, ClaI, PstI, and NdeI. (The XbaI digest was only 50% complete.) We then transcribed these substrates in the presence of [a-”PICTP and separated the products on a denaturing 4% acrylamide, 7 M urea gel (Fig. 4B). This experiment demonstrates a doublet of transcripts (designated by dots) which are progessively shortened as the SalI fragment is cleaved with these restric- tion enzymes. We interpret these RNAs as run-off transcripts which initiate within the T4 DNA and proceed to the end of the template (in each case defined by the position of the restriction site). By comparing the restriction map (Fig. lA) with the sizes of these RNAs, we estimate that these tran- scripts initiate from two promoters which lie 500 and 600 bp upstream of the NdeI site. Initiation at these promoters generates transcripts that extend in vitro through the region we have assigned to the gene for the 46-kDa protein.

The 46-kDa Protein Is a T4 dsDNA-binding Protein, Absent after Infection of a T4 UUSX- Phage-The region we have assigned to the 46-kDa gene lies in a part of the T4 genome which is transcribed early after infection in vivo and which contains several genes for proteins that interact with DNA (2). To test whether the 46-kDa protein might also have an affinity for DNA, we examined the dsDNA cellulose-binding proteins expressed by DH1 cells containing either the vector pKG1810R3, the plasmid pDH447, or infected with T4 41- phage (Fig. 5, left panel). The 46-kDa protein expressed by pDH447 binds to the dsDNA cellulose in the presence of 0.1 M NaCl, is eluted by stepping the salt concentration to 0.5 M NaCl (lune E), and co-migrates with a similarly bound protein present in fractions from extracts of T4 41”infected cells. As

Cloning of T4 uvsX Gene 5667

9420

6560 - 4360 -

2320 - 2025 -

565 -

A

1 2

1900 - 1375 - 1580 -

950 - 830 -

565 -

I 2 3 4 5 6

FIG. 4. In vitro transcription of T4 DNA restriction frag- ments demonstrates two transcripts initiating upstream of the gene for the 46-kDa protein. T4 restriction fragments were tran- scribed in uitro as described under “Experimental Procedures” and in the text. Panel A , RNA products were obtained after transcription of (lane I ) the 4330-bp SalI fragment (map units 24.8 to 20.75) or (lane 2) the 3400-bp EcoRI fragment (map units 24.3 to 21.15) in the presence of [ W ~ ~ P ] U T P and displayed on a 1% nondenaturing agarose gel. The positions of heat-denatured 32P-labeled HindIII fragments of X DNA are indicated. Note that the sizes of the RNAs cannot be determined from this gel because the gel is nondenaturing. Panel B, denaturing 4% polyacrylamide, 7 M urea gel showing RNA products (labeled by [a-”P]CTP) obtained after transcription of the above SalI fragment (lane I ) or the fragment digested with BglII (lane 2), XbaI (lane 3) , ClaI (lane 4 ) , PstI (lane 5), or NdeI (lane 6). (The XbaI digestion was 50% complete). The positions of size markers obtained by digesting X DNA with HindIII and EcoRI are shown. The doublet of transcripts observed after digestion of the SalI fragment with the various restriction enzymes (see text) is indicated by dots. We calcu- late the sites of these transcripts as follows: 1800 and 1690 bases (lane 3); 1320 and 1200 bases (lane 4) ; 880 and 780 bases (lane 5); and 600 and 500 bases (lane 6).

expected, no such protein is expressed by cells containing the vector.

Previously, the genes for two T4 proteins which interact with DNA have been genetically mapped upstream of gene 41. One of these genes encodes P-glucosyltransferase, a protein of 46,000 daltons (47) which adds monoglucosyl moieties to DNA (reviewed in Ref. 48). We find no P-glucosyltransferase activity (see “Experimental Procedures”) in crude extracts of cells with plasmids expressing the 46-kDa protein or in the dsDNA cellulose-binding fraction of pDH447/DH1 cells (data not shown). The other T4 protein which interacts with DNA and whose gene has been mapped upstream of gene 41 is

uvsX. As indicated in the Introduction, this protein has been shown to be analogous to the E. coli recA protein in promoting ATP-dependent homologous pairing in vitro (26-29). We have identified the 46-kDa dsDNA-binding protein present in cells with pDH447 or after infection with wild type T4D as the uvsX gene product, since this protein is not found after infection with the T4 uvsX- mutant, fdsA (Fig. 5 , rightpanel, lane E ) or with T4 UVSX am1 1 (not shown). Previous workers (26, 28, 49) have estimated the size of the uvsX protein as approximately 40 kDa, in contrast to its apparent size of 46 kDa on our gels. This discrepancy is due to the conditions of gel electrophoresis since the uvsX protein made by pDH447 and that purified from T4-infected cells (the generous gift of T. Formosa, University of California, San Francisco) migrate as 46 kDa under our conditions (data not shown) and as 40 kDa under conditions (50) similar to those used by Formosa and Alberk3s4 The reason for the anomalous electrophoretic behavior of the uvsX protein is not clear. Although for con- venience we have referred to the uvsX protein as the 46-kDa band on protein gels, its actual size will have to be determined by other techniques.

The uusX Protein Expressed by the Recombinant Plasmids Complements T4 uvsX- Phage-The phenotype of T4 uvsX- mutants is pleiotropic including increased sensitivity to UV light, ionizing radiation, and chemical agents, as well as arrest of DNA synthesis and decreased burst size (see Introduction). In collaboration with J. W. Drake (National Institute of Environmental Health Sciences), we tested the biological activity of the uvsX protein expressed by our clones by asking whether UV-irradiated T4 uvsX- phage have increased sur- vival in cells containing the 46-kDa protein. T4x, (uusX-) phage were irradiated and then plated on DH1 cells without plasmid, with the vector, or with pDH447. As controls, wild type T4D or T4 deficient in two other genes associated with recombination and repair, uvsY and denV, were also irradi- ated and plated. Each control phage has a distinctive survival curve which is not altered by the host (Fig. 6, solid symbols). However, after 83 s of irradiation, the irradiated uvsX- phage survive approximately 8-fold better (to a level nearly like that of T4D) when plated on the pDH447/DH1 cells (open trian- gles) than on DH1 or DH1 with the vector (open circles and squares). This experiment indicates that the uvsX protein expressed by the clone can substitute for the T4 uvsX gene product in vivo in responding to damage by UV light.

As discussed in the Introduction, T4 uvsX- mutants have been shown to suppress conditionally lethal mutations in the T4 gene 49 (22-24). Thus, at the nonpermissive temperature, T4 uvsX- ts gene 49 phage will grow but T4 ts gene 49 phage will not. We asked whether providing uvsX protein from the uvsX+ plasmid pDH428 could reduce this suppression. As seen in Table I, use of the pDH4281DHl host lowers the efficiency of plating of the T4 fdsA (uvsX-)-tsCS (ts gene 49) phage at the nonpermissive temperature by greater than 20-fold. The use of cells containing the vector or the plasmid pDH428A3 (with the 800-bp deletion which eliminates the 46-kDa product) does not affect the efficiency of plating. In addition, the ts gene 49 mutation by itself displays a tighter phenotype on pDH428/DH1 cells than on cells that do not express uvsX protein. Taken together, the results of these complementation experiments indicate that the uvsX protein expressed by our clones behaves like a biologically active uvsX gene product.

Purification of the uvsX Protein from pDH4471DHl Cells- The purification of the uvsX protein (see “Experimental

T. Formosa, personal communication. ‘ D. M. Hinton and N. G. Nossal, unpublished results.

Cloning of T4 uvsX Gene 5668

68 -

43 -

I VECTOR I pDH447 I T4=N81(41-) I A B C D E F A B C D E F

. " . U . ' L " . . ~ ' I ' I

46-kDa pro te in-

- 68

- 43

FIG. 5. The 46-kDa protein is a dsDNA-binding protein, copurifying with a protein present after infection by T4 phage but absent after infection by T4 with the fdsA mutation in the UVSX gene. Cell lysates were partially purified by chromatography on dsDNA cellulose columns as described under "Experimental Procedures" and the proteins displayed on 10% (left) or 12.5% (right) polyacrylamide-SDS gels. For each extract, lane A represents 5 pl (left gel) or 3 pl (right gel) of the dialyzed 130,000 X g supernatant; lane B, 5 p1 (left gel) or 3 pl (right gel) of the proteins flowing through the column; and lanes C-F, 15 p1 (left gel) or 20 pl (right gel) of the proteins eluting with DC buffer plus 0.05, 0.1, 0.5, and 1.0 M NaCl, respectively. The positions of the 46-kDa

5 protein as well as protein standards having molecular weights of 68,000 (bovine serum albumin) and 43,000 (ovalbumin) are indicated. The left gel shows extracts from DH1 cells containing the vector (pKG1810R3), the plasmid pDH447, or infected with T4arnN81 (gene 41). The right gel shows extracts from DH1 or pDH447/DH1 cells infected with T4D or T4 fdsA (UUSX-) .

Procedures") is summarized in Table I 1 and Fig. 7. Most of the purification is achieved by chromatography of the crude extract over dsDNA cellulose, which yields a fraction contain- ing the uvsX protein as the major protein (Fig. 7, lane C). The remaining DEAE and phosphocellulose steps are modi- fications of procedures developed for the purification of uvsX protein from T4-infected cells by Formosa (27-29).3 During the last chromatographic step (phosphocellulose column) uvsX protein, apparently homogeneous by gel electrophoresis, co-chromatographs with a ssDNA-dependent ATPase activity (Fig. 7) which gives both ADP and AMP as products (see Footnote e to Table 11). The purified protein is free of nu- cleases acting on single-stranded, double-stranded, or super- coiled DNA under the conditions used for the strand exchange reactions below (Figs. 8 and 9).

Strand Exchange Catalyzed by the uusX Protein in Vitro- We have verified that the uvsX protein from the clone cata- lyzes D loop formation (data not shown) and strand exchange between circular ssDNA and homologous dsDNA (Figs. 8 and 9), as originally reported for the uvsX protein from T4- infected cells (26-29). Fig. 8 shows the products of strand exchange between single-stranded circular viral 4x174 DNA and double-stranded 4x174 RF linearized by cutting with PstI nuclease. (The PstI digestion gives a 4-base single- stranded extension at the 3' terminus.) At the lower concen- tration of ssDNA, strand exchange is catalyzed by the uvsX protein alone (Fig. 8, lane 5 ) but is strongly stimulated by the T4 gene 32 helix-destabilizing protein (lane 4 ) . At the higher ssDNA concentration, both the uvsX and 32 proteins are apparently required (Fig. 8, lane I ) , since there are no visible

products with only the uvsX protein (lune 2) or 32 protein (not shown). A portion of the products in lanes 1 , 4 , and 5 co- migrate with marker nicked 4X RF 11, the expected product of complete strand exchange. Since we have not further char- acterized these products, it is possible that products other than simple nicked circles co-migrate with the nicked circle marker. There are also products migrating behind the nicked circle marker, which may be intermediates in which the linear duplex is only partially unwound. Products running behind the nicked circle are also seen in similar reactions catalyzed by recA protein (51). In Fig. 8, there are, in addition, large products which do not enter the gel and which are more prominent at higher ratios of dsDNAssDNA (lanes 4 and 5 versus lane I). These may be complex structures resulting from incomplete strand exchange involving several molecules.

Fig. 9 demonstrates that the strand exchange reaction cat- alyzed by the uvsX protein alone is also stimulated by increas- ing the concentration of dsDNA. As described under "Exper- imental Procedures," we labeled the 5224-bp XhoIIPstI frag- ment of 6x174 RF DNA at the 3' terminus of the (-)-strand by adding nucleotides to the 3' OH of the XhoI end using the large fragment of E. coli polymerase I to give blunt ends. At the lower dsDNA concentration, the uvsX protein alone does not yield visible products (lane 4 ) . However, raising the dsDNA concentration 2-fold allows a reaction by the uvsX protein alone (compare lanes 7 and 4). The addition of 32 protein (Fig. 9, lanes 3 and 6) increases the extent of the reaction mediated by uvsX protein at both concentrations of dsDNA. Note that products co-migrating with nicked circular DNA and presumably representing extensive branch migra-

Cloning of T4 uvsX Gene 5669

I 2'0 4'0

. I 60 8 0 160 Id0

UV Dose (sec)

FIG. 6. The presence of pDH447 (uusX' plasmid) specifi- cally increases the survival of UV-irradiated T4 UUSX- phage. T4D, T4 fdsR (UUSY-) , T4ul (denb"), and T4x, (UUSX-) were irra- diated for the indicated times a t a UV dose rate of 0.3 J m-* s-l and plated on DH1 without plasmid (0, 0), pKG1810R3 (vector, M, O), or pDH447/DH1 (uvsX+) plasmid, A, A). The uusX- phage is indi- cated by open symbols; all other phage are shown with closed symbols. The surviving fraction represents the titer obtained after irradiation relative to that prior to UV treatment.

TABLE I Suppression of a T4 49- mutation by a UUSX- mutation is eliminated

in cells with a uvsX+ Dlnsmid Efficiency of plating

44 TI29 "c" uvsx

pKG1810R3/DHl No 0.95 1.11 0.23' 0.78 pDH428/DHl Yes 0.99 0.90 C0.003 0.04 ~DH42863/DHl No 0.81 0.93 0.22' 0.98 "The T4 phage were plated as described under "Experimental

Procedures." The efficiency of plating 44 "C/29 "C represents the titer of the phage at the nonpermissive temperature (44 "C) relative to that at the permissive temperature (29 "C). ' Plaques formed at 44 "C were very small.

tion following synapsis are much more prominent in the presence of 32 protein.

DISCUSSION

Cloning of the Bacteriophage T4 Gene uvsX and Preliminary Characterization of the uvsX Protein Expressed by the Clone- The bacteriophage T4 uvsX gene is a nonessential gene whose product, along with other proteins, is required for wild type resistance to radiation and chemical treatment, normal re- combination frequencies, and the formation of a network of T4 DNA thought to arise through homologous recombination

TABLE I1 Purification of the T4 uvsX protein from pDH447lDHl cells

ATP hydrolyzed Protein UVSX protein"

mg mglml mg amollmg I. Dialyzed crude 1650' 33

11. dsDNA cellulose 44 3.7 111. DEAE-cellulose 9.4' 1.5 9.4c.d 6.8"' IV. Phosphocellulose 5.3' 2.1 5.3' 7.6'

' From 20.5 g of cell paste. e Calculated as if total extract purified through each step.

extract

Identified by SDS-gel electrophoresis.

This is uvsX protein in peak fractions used for phosphocellulose chromatography. Total uvsX protein recovered from DEAE column (corrected as in Footnote c) was 17 mg.

'DNA-dependent ATPase cannot be measured reliably in earlier fractions. In both fractions I11 and IV, the ratio of AMP to ADP was 0.28, and there was no ATP hydrolysis in the absence of ssDNA.

FIG. 7. Polyacrylamide-SDS gel electrophoresis and ssDNA-dependent ATPase activity of phosphocellulose frac- tions of the uvsX protein. Lane A , bovine serum albumin ( M , = 68,000) and ovalbumin ( M , = 43,000) marker proteins; lane B, di- alyzed crude extract (2 pl); lune C, dsDNA cellulose-pooled enzyme (5 pl) ; lane D, DEAE-cellulose-pooled enzyme (5 pl); and lunes 2-26, 5 pl of the corresponding even-numbered phosphocellulose fractions. The 10% polyacrylamide gel was stained with Coomassie Blue. Single- stranded 6x174 DNA-dependent ATPase was measured with 0.5 p1 of the indicated fractions. (Dashed line is NaCl concentration.)

among replicating T4 genomes (see Introduction). We have found that plasmids containing the T4 DNA approximately 300-2900 bp upstream of gene 41 express a 46-kDa protein whose presence both imparts increased survival to UV-irra- diated T4 uvsX- phage (Fig. 6) and decreases the suppression of a conditionally lethal T4 ts gene 49 mutant by a T4 uvsX- mutant (Table I). These in vivo results are consistent with our having cloned the gene for a biologically active uvsX protein, and our position for the UVSX gene agrees with genetic mapping of the uvsX gene between T4 genes 41 and 42 (25, 30). The T4 uvsX+ plasmids described here, together with the T4 uvsY+ and uvsW+ plasmids previously described (19, 52),

5670 Cloning of T4 uvsX Gene

ssDNA

uvsx

32

nc

dsl

ssc

i.8 AJM 2.9 uh T” - ‘ 2 3 I+ 4 5 6

FIG. 8. T4 uvsX protein catalyzes strand exchange between single-stranded 4x174 circular DNA and linear duplex 6x174 RF DNA in a reaction stimulated by T4 32 protein. Reaction mixtures contained no proteins (lunes 3 and 6), uvsX protein (412 pg/ml) alone (lunes 2 and 5 ) , or uvsX protein plus T4 32 protein (37 pg/ml in lunes 1 and 4 ) . 6x174 RF 111 DNA (cut by PstI nuclease) was 17 p~ (nucleotide), and ssDNA concentration was either 5.8 PM (nucleotide) in lunes 1-3 or 2.9 PM in lunes 4-6. The positions of 4x174 single-stranded circular DNA (ssc), double-stranded linear DNA (dsl), and nicked circle RF I1 DNA (nc) markers are indicated.

dsDNA

ci

FIG. 9. Increasing the linear dsDNA concentration facili- tates strand exchange with circular ssDNA by the uvsX pro- tein in the absence of 32 protein. Lanes 1-5 and lanes 6-8 contained the blunt end 5224-bp [3ZP]Xho-Pst fragment of 4x174 RF DNA (see “Experimental Procedures”) at final concentrations of approximately 1.4 and 2.8 p~ (nucleotide), respectively. All reactions contained 2.3 p~ (nucleotide) single-stranded 6x174 viral DNA and, where indicated, 412 pg/ml uvsX protein and 110 pg/ml 32 protein. Lanes 5 and 8, proteins omitted; lanes 4 and 7, uvsX protein; lanes 3 and 6, uvsX and 32 proteins; lane 2,32 protein; and lane 1 , uvsX and 32 proteins without ATP. The positions of 6x174 nicked circle RF I1 DNA (nc) and double-stranded linear DNA (dsl) markers are indicated.

should aid in our understanding the role of each of these genes in T4 DNA recombination and repair.

The uvsX protein that we have purified from plasmid- containing cells is a ssDNA-dependent ATPase which pro- motes homologous strand exchange in vitro, as has been previously shown for uvsX protein from T4-infected cells (26- 29). Our finding that the plasmid-encoded uvsX protein hy- drolyzes ATP to both ADP and AMP (Table 11) is in agree- ment with the more extensive analysis of the products formed by the uvsX protein from infected cells (27-29) and eliminates the possibility that one of these products was produced by a contaminating phage protein. uvsX protein from cells with the plasmid mediates ATP-dependent strand exchange be- tween the (-)-strand of a linear duplex and its single-stranded circular (+)-strand complement in a reaction which is stimu- lated by, but not absolutely dependent upon, the T4 gene 32 helix-destabilizing protein (Figs. 8 and 9). Strand exchange in the absence of 32 protein is highly dependent on the concentration of both ssDNA and dsDNA (Figs. 8 and 9), which may explain previous reports of a 32 protein require- ment for the strand exchange reaction (cited in Ref. 29). However, we cannot rule out the possibility of a fundamental difference between the uvsX protein from the clone and that from T4-infected cells. Our results, indicating a stimulation but not a requirement for 32 protein in uvsX protein-mediated strand exchange, resemble those previously observed for the E. coli ssDNA binding (SSB) protein and the E. coli recom- bination protein recA ((51); for a recent review, see Ref. 53). More detailed investigations of the T4 recombination proteins will be required in order to understand precisely how homol- ogous recombination is accomplished by purified T4 proteins in vitro as well as during the life cycle of T4.

Expression of the uvsX Protein-Our plasmids which con- tain the T4 uvsX gene cloned in a multicopy plasmid vector express a high level of the protein both in vivo (Fig. 2) and in vitro (Fig. 3). We have presented evidence that at least part of this expression is due to transcription promoter(s) on the T4 DNA upstream of the uvsX gene. First, expression of the protein is independent of the orientation of the T4 DNA within the plasmid (Fig. 2), suggesting that in the plasmid the uvsX gene is not expressed simply by read-through transcrip- tion from the vector DNA. Second, our in vitro transcription of T4 DNA fragments from this region identifies 2 tandem promoters lying upstream of the uvsX gene which produce transcripts that extend through the uvsX gene (Fig. 4). We have observed transcripts from these promoters under con- ditions (see “Experimental Procedures” and “Results”) which should favor specific initiation from stronger promoters over nonspecific starts or initiation a t weaker promoters (54). A previous study by Gram et al. (55) to identify strong T4 promoters used in vitro by E. coli RNA polymerase failed to reveal the ones we have observed upstream of the uvsX gene. This discrepancy probably reflects the difference in salt concentrations used in their study (200 mM KCl) and our study) (75 mM) since we find that the intensity of the RNA bands observed after transcription of the T4 Sal1 fragment (map units 23.8 to 20.75) is diminished severalfold using the higher sal t c~ncentrat ion.~ However, even at the higher salt concentration, the initiation of transcription is very specific, yielding product which comigrates with the major transcrip- tion band observed in Fig. 4A, lane 1.

I n vitro transcription/translation of the plasmid with the largest T4 DNA insert, pDH428, produces several minor species (Fig. 3) in addition to the uvsX protein. Longer

D. M. Hinton, unpublished experiments.

Cloning of T4 uvsX Gene 5671

exposures of this autoradiogram reveal that two of the fainter of these bands co-migrate with either p-lactarnase, the amp' gene product observed with the vector alone, or with T4 gene 41 protein, which we have previously shown is expressed by pDH428 in uiuo (31). Most of the remainder of the bands migrate faster than the 46-kDa UVSX product and are depend- ent on the T4 DNA for expression. These minor bands could arise from premature transcription or translation stops of the highly expressed uvsX gene or could be unidentified T4 pro- teins. In fact, T4 gene 40, whose product, a 14-kDa protein, is required for proper phage head assembly has been mapped, like the uvsX gene, between genes 41 and 42 and is transcribed in the same direction as gene 41 (56). Whether one of the minor protein bands we have observed after translation of the uvsX plasmids is this gene product is not yet known.

We are presently examining the in vivo expression of the T4 genes cloned in pDH428. If the promoters we have detected i n vitro are active on the T4 genome, they should be recognized early after T4 infection before the extensive T4 modifications to E. coli RNA polymerase have altered the polymerase's promoter recognition (reviewed in Ref. 57). Both uvsX and 41 are prereplicative genes expressed within the first 5 min of infection (49), but it is not known if this expression results from the promoters we have observed in vitro. In addition, it is unclear why the level of gene 41 protein expressed by pDH428 in vivo and in vitro is strikingly less than that of the uvsX protein (Figs. 2 and 3). We are investigating whether this difference in the levels of uvsX protein and 41 protein expressed by the plasmid is due to a regulatory mechanism and, in addition, if it mimics the expression of these proteins on the T4 genome.

Acknowledgments-We thank T. Mattson (University of Geneva, Switzerland) for the plasmid pVH627 and M. Nakinishi and B. Alberts (University of California, San Francisco) for showing us their T4 DNA sequence analyses prior to publication. We are especially grateful to J. Drake (National Institute of Environmental Health Sciences, Research Triangle Park, NC) for phage and the use of his laboratory for the UV sensitivity experiment and to T. Formosa (University of California, San Francisco) for sending us purified uvsX protein and personal communications. We also thank David Rogerson for fermentor growth of plasmid-containing cells, Ross Richardson, Edith Miles, and Howard Nash for helpful discussions, and Helen Jenerick for typing this manuscript.

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Continued on next page.

5672 Cloning of T4 UVSX Gene

SUPPLEMENTARY MATERIAL TO

AND PURIFICATION AND CHARACTERIZATION OF THE T4 uvsX RECOMBINATION PROTEIN CLONING OF THE BACTEERIOPHAGF T4 GENE

Deborah M. Hinton and Nancy G. Nossal Section On Nucleic Acid Biochemistry Laboratory of Biochemical Pharmacology

National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases National Institutes of Health

Bethesda, Maryland 20892 Building 4, Room 116

EXPERIMENTAL PROCEDURES Unless otherwise indicated, materials and methods were those described

Bacterial Strains, Phage, and Plasmids - E. coli DH1, a Su+ recA-

strain, has been described ( 3 3 ) . T4 fdsA (uvSX-) 122, 23), T4 fdsB ( 6 1 (22, 231, T4 tsCB 1% gene 49) (221, T4 fdsA tsC9 122). T4vl (20, 3 0 , T4& (&) Ill), and T 4 s (d) (24) were obtained from J. W. Drake (Nstional Institute of Environmental Health Sciences). T4amN81 (gene

previously 131. 32).

41) (3) was from W. B . Wood (University of Coloradol. The procedures used for constructing plasmids and the construction of

the plasmids pLS501, pLS502, pDH428, and pDH4112 have been described 1311. In summary, pDH428 (Figs. 1A and 1El was constructed by inserting the 3400 bp

- EcoRI fragment representing T4 map units 24.3 to 21.15 [isolated from pVH627 (35, 36)l upstream of the T4 DNA (map Units 21.15 to 20.06, e R 1 to edIIIl in pLS501. Plasmid pDH421 (Fig. 1A1, another product Of that ligation re- action, contains the E R I fragment in the opposite orientation. In all of

pBR322-based gall('expression plasmid designed by McKenney and Rosenberg (37). these plasmids, the vector DNA is a ~ R I / ~ d I I I fragment of pKG1810, a

The vector DNA is schematically shown as the non-T4 DNA in Fig. 1 B . It con- tains the IS2 transcription termination site upstream of the galK gene.

The pDH428 deletions (Fig. 1A) were constructed as follows. For A1 and

NdeI. The resulting DNA was treated with T4 DNA polymerase in the presence d 5 , pDH428 was digested to completion with S I 1 and partially digested with

Of the four dNTPS (31) to produce blunt end DNA. pDH428A1 and pDH428A5 represent two plasmids isolated after ligation Of this DNA and transformation of DH1 (38). [Note that the deletion in pDH42861 extends from the S I 1 Site in the T4 DNA through the e 1 Site in the vector (Fig. 1B).l A similar procedure was used to Construct pDH428A2 (after *I x S I 1 digestion) and pDR428A4 (after G I x -1 digestion). [The *I site was predicted by DNA

comunication).l pDH428A3 resulted from digestion of pDH428 with G I fol- sequence analysis of this region by M. Nakanishi and B. Alberts (personal

lowed by ligation (no T4 DNA polymerase treatment).

-

from pVH627 was ligated to the e R I / ~ d I I I vector fragment of pKGl8lO TO Construct pDH447 and pDH4426 (Fig. 1Al. the 3400 bp g R I fragment

produce blunt end DNA at the remaining G d I I I and =HI termini) and ligated (described above). The products were treated with T4 DNA polymerase (to

again. pDH447 and pDH4426, two products of this procedure, represent inser- tions of the 3400 bp X R I fragment in opposite orientations.

To Construct the control plasmid pKGl8lOR3, the KRI/&dIII vector

presence of the &HI OligOdeOxyribonuCleOtide linker pd(GGAATTCC). fragment of pKGl8lO was treated with T4 DNA polymerase and ligated in the

In Vitro Transcr ipt ion/Translat ion Reactions - Plasmid DNA, isolated as

bromide-cesium chloride gradients 1391. After removal of the ethidium bro- described (311, was purified by twice centrifuging to equilibrium in ethidium

mide by extraction with CsC1-saturated isopropanol, the DNA Solution was dialyzed against TE buffer ( 1 0 mM Tris-HCI, pH 7.9, 1 mM EDTA), and the DNA precipitated by the addition of salt and ethanol. The DNA pellet was washed with 70% ethanol, dried, and redissolved in TE buffer to give a final concen- tration of 0.2 to 1.5 mg/ml. The DNA Was stored at -2O'C.

(amount given in legend to Fig. 3). 6 111 of E. coli I~CB', recc- 5-30 extract " In vitro t r a n s c r i p t i o n / t r a n s l a t i o n reactions (10 "11 contained DNA

sium acetate, 2 mM =ATP, 0 . 5 mM each rCTP, rUTP, =GTP, 0.2 mg/ml (Worthington), 30 mM Tris-HC1, pH 8.0. 120 mM potassium acetate, 15 mM magne-

kinase, 10 "M each amino acid except methionine. and 1.1 yM [35slmethionine tRNA, 0.1 mM cyclic AMP, 1 mM phosphoenolpyruvate, 0.01 mg/ml pyruvate

nine (110 pmol, 1 rl) was added, and the reaction incubated for 10 minutes. (680 to 980 Ci/mol). After incubation at 37OC for 40 minutes, cold methio-

Reactions were Stopped by adding 100 ill gel loading solution 19% glycerol, 2% Sodium dodecyl sulfate (SDS), 57 mM Iris-HCL, pH 6.8. 4.8% 2-mercaptaethanol, and 0.05% bromophenol blue] and placing the tubes in a boiling Water bath for 2 minutes. The proteins in aliquots representing 0.7 to 1.4 u1 of the origi- nal reaction mixture were separated on polyacrylamide, SDS gels as described (31). Before autoradiography, gels were fixed by shaking in 10% TCA fox 20 minutes, treated with en3Hance (New England Nuclear Corp.), and dried.

In Vitro Transcription Reactions - The T4 e 1 restriction fragment, representing map Units 24.8 to 20.75 (Fig. 1A) and isolated from T4 dC-DNA, and the T4 e n 1 fragment representing map units 24.3 to 21.15 (Fig. 1A) and isolated from the plasmid pVH627 (see above) were purified a5 described (31). or the experiments shown in Fig. 4 ~ . the purified SI fragment was incubat- ed with each of the restriction enzymes indicated. After digestion. the reaction mixtures were extracted with phenol and the DNA precipitated by the addition of Salt and ethanol, washed with 70% ethanol, dried, and redissolved in TE buffer. DNA fragments were transcribed essentially as described (40) in reaction mixtures ( 1 5 ul) containing 20 mM Tris-HC1. pH 7.9, 6% glycerol, 75 mM KCl, 5 mM MgC12, 0.1 mM EDTA, 0.5 mM dithiothreitol, 50 ug/ml bovine serum albumin, 0.01 pmol DNA fragment(s1, 0.02 pmol E- a RNA polymerase

phates IrNTPs). The reaction mixture containing all components except hepa- (Enzo Eiolabs), 100 ug/ml heparin, and all four 5'-ribanucleaside triphos-

rin and the rNTPS was first incubated at 37-c for IO minutes. Heparin was

then added and the resulting mixture kept at 37'c for 2 minutes. In vitro transcription was begun by adding a solution of either 10 VM (,-3Zp)~~p ( 6 0

rATP and rGTP, 50 uM rCTP or rUTPl. Cold ribonucleoside triphosphates, 1 mM Ci/TUlOl) Or 10 UM IO-~~PICTP (82 ci/mmoll plus the other three rNTPS (200 uM

rNTP mixture in Order to reduce transcription pausing. After an additional each =ATP. rCTP, rUTP. and =GTP, were added 10 minutes after the radioactive

10 minutes, a solution (155 "1) containing 77 mM Tris-HC1, pH 7.9, 52 m~

MgC12, 0.4% SDS, and 150 uglml E . coli tRNA was added and the reaction stopped by phenol extraction and ethanol precipitation. The RNA products Were then either (il dissolved in 4 mM EDTA, pH 7.6, 0.02% bromophenol blue,

Seconds, quick-cooled on ice, and electrophoresed on a 1% agarose gel in 1 x

0.04% xylene cyanol FF, and 12 % glycerol, heated in boiling water for 30

TBE buffer plus 7 M urea, 0.1% bromophenol blue, and 0.1% xylene cyanol FF, TBF buffer I89 mM Tris-borate, pH 8.3, 2.5 mM EDTA1 or (ii) dissolved in 1 x

heated at 70-C for 3 minutes, and electrophoresed on a 4% aorylamide, 7 M urea gel in 1/2 x TBE buffer.

Complementation of T4 uvsx- Phage by Infection of Cells Containinq uvsX+ Plasmids - TO test for the elimination of the T4 phage Suppression of T4 49- phage by the presence of a uysX+ plasmid, wild type T4D. T4 fdsA I&), T4 tSC9 (ts gene 49), or T4 fdsA-tsC9 phage were plated as described (24) on DH1 cells or DH1 cells containing the indicated plasmids except that phage were not preabsorbed before plating. Plates were incubated overnight at 29°C (permissive for T4 tsC9 growth) or 44'C (nonpermissive for T4 e). The efficiency of plating I-epresents the titer at the nonpermissive tempera- ture relative to that at the permissive temperature.

with a uysx+ plasmid, T4 phage were irradiated and plated essentially as

To test far increased survival of irradiated T4 phage in a host

described (24). T4 phage at 2 to 3 x 10' phage/ml in M9S medium (241 Were fixst irradiated for the indicated times using a 15-watt low pressure Hg bulb

diluted in D broth (24) and plated (without a preadsorption step) in dupli- (uv dose rate of 0.3 J m-2 sec'l). Irradiated phage were then appropriately

care on DH1, DH1 with a uysx+ plasmid (pDH447). or DHI with the VeCtOr (pKG1810R3). Phage were plated under dim yellow light to avoid the possibil-

the number of plaques observed for the irradiated phage relative to the ity of photoreactivation repair. The surviving fraction (Fig. 6 ) represents

number of plaques obtained for nonirradiated phage on a given host. Fractionation of Extracts from uvsx' and UVSX- Cells by Double-Stranded

DNA Cellulose Chromatograph1 - DH1 cells with or without plasmid were grown at 37-C in L broth (31) or L broth plus 25 ugh1 ampicillin (for cells con- taining plasmids) to a concentration of 2 x 10' cells/ml. For T4 infection, T4 phage (1 x were added to 200 ml of culture and the in- fected culture incubated 12 minutes (for T4D or T4 fdsA phage) or 40 minutes [for T4- (gene 41) phage]. Both infected and uninfected Cultures were then poured onto 100 ml frozen L broth and the cells collected by centrifuga- tion. The cell pellet was resuspended in 3 ml sonication buffer (40 mM

Tris-HC1, pH 8.0. 10 mM MgC12, 2 mM CaCl2. 1 mM EDTA, 100 mN NaC1, 10% glycer- ol, and 1 mM 2-mercaptoethanol) and the cells broken by sonication. The resulting extracts were incubated at 15'C for 45 minutes with 25 ug pancrea- tic DNase (Worthington DPFF, RNase Free) and then centrifuged for 90 minutes at 40,000 rpm in a Beckman 50 Ti rotor. The 130,000 x g supernatants Were

mM 2-mercatoethanol, 10% glycerol) plus 5 0 mM NaC1. Aliquot3 (0.6 mll Were

dialyzed extensively against DC buffer I20 mM TriS-HC1, pH 8.0, 1 mN EDTA, 1

equilibrated in the same buffer. After the columns were washed with 0.5 ml applied to double-stranded calf thymus DNA cellulose columns ( 0 . 1 mll (41)

steps of 0.2 ml DC buffer with 0.1 M NaC1, 0.15 ml DC buffer with 0.5 M NaC1, of the 0.05 M NaCl buffer (abovel, the d5DNA binding proteins were eluted by

and 0.15 ml DC buffer with 1 . 0 M NaC1. The eluates were Stored at -7O'C. Purification of T4 uysx Protein - a DHl/pDH447 was grown in a

50-liter fermentor to late log phase at 37OC in L broth plus 25 pg/ml ampi- cillin, The cell paste (129 g) was stored at -7OOC.

Extract - 20.5 g of cell paste were thawed at 4°C in 60 ml of the sonication buffer described above supplemented with 1 mM phenylmethylsulfonyl fluoride IPMSF). The cells were sonicated in SO-second pulses, keeping the

___

temperature below 8OC with a salt ice bath, until the optical density at h

concentration of 17 pg/ml, and the extract incubated for 60 minutes at 15°C 540 had decreased from 1.6 to 0.3. Pancreatic DNase was added to a final

and centrifuged for 2 hours at 35,000 rpm in a Beckman 45 Ti rotor. The supernatant solution (50 ml) was dialyzed over a period of 20 hours against 4 changes of 1 liter of DC buffer with 5 0 mM NaCl and 1 mM PMSF.

stranded calf thymus DNA cellulose was washed with 165 ml of DC buffer with dsDNA Cellulase Chromatography - A 1.5 m x 11 cm column of double-

50 mM NaCl until no more material absorbing at 260 mu was eluted. The dia- lyzed crude extract was loaded at 10 ml/hour. The column Was washed with 200 rnl of DC buffer with 50 NaC1, 84 ml of DC buffer with 0.1 M NaCl, and a linear gradient formed from 150 ml of DC buffer with 0 . 1 M NaCl to 145 ml of DC buffer with 0.8 M NaC1. The uvsX protein, monitored by SDS gel eleCtr0- phoresis, was eluted at 0.2 M NaCl in a volume of 12 ml.

cellulose fractions (4.8 ml total1 was dialyzed against 3 changes of 600 ml DEAE-CellUloSe Chromatography - Eighty percent of the three peak dSDNA

of buffer A (20 mM Tris-C1, pH 7.4, 1 m 8-mercaptoethanol, 0.2 mM EDTA, and 10% glycerol) with 0.1 M NaCl over 26 hours and loaded at 5 ml/hour Onto a

The column was washed with 10 ml of this buffer, and the protein eluted with 5-ml column of Dm-Sephacel (Pharmacia), equilibrated in the Same buffer.

buffer A with 0.45 M NaC1. The UVSX protein (essentially homogeneous) was

a linear gradient formed from 50 ml of buffer A with 0.1 M NaCl to 50 ml of

eluted at 0.16 M NaCl in a total of 11 ml.

Cloning of T4 uvsX Gene 5673

Phosphocellulose chromatography - Fifty percent of the five peak frac- tions 12.5 ml total) from the DEAE column was diluted with an equal volume of buffer A and loaded at 4 mlfhour Onto a 2-ml column of Whatman P-11 phospho- cellulose which had been equilibrated with buffer A with 0.1 M NaC1. The column was washed with 4 ml of buffer A with 0.1 M NaCl and then with a linear gradient of 0.1 to 0.8 M NaCl in buffer A I40 ml total). Greater than 90% of essentially homogeneous UVEX protein was eluted in a single 1-ml fraction at 0.15 M NaC1.

pH 7.5, 5 mM MqClZ, 100 ilg/ml bovine serum albumin, 10 mM dithiothreitol, 58

UVSX ATPase Assax - Reaction mixtures (3 ul) containing 50 mM Tris-C1,

YM (nucleotide) 0x174 ssDNA, 1 mM [14ClATP (3 cpmlpmol). and enzyme were

chromatography on PEI thin layers (Brinkman) in 1.2 M LiC1, and the appropri- incubated for 30 minutes at 37'C. The nucleotide products were separated hy

ate areas counted in Bioflor scintillation solution (New England Nuclear).

RF I Were prepared as described ( 4 2 ) . Linear double-stranded 0x174 labeled Strand Exchange Reactions - Single-stranded 0x174 viral DNA and 0x174

at the 3' OH end of the ( -1 strand was prepared by digesting 0x174 RF I DNA with =I and =I nucleases and then adding nucleotides to the 3'OH of the

England Biolabsj in the presence of dCTP, dGTP, dTl'P, and [''PldATp as de- Scrihed (43), giving 500 cpm/pmol DNA nucleotide. Strand exchange reaction mixtures contained 50 mM Tris-C1, pH 7.5, 5 mN MgClZ, 10 mM dithiothreitol, 100 eg/ml bovine serum albumin, 5 mM ATP, 4 mM phosphocreatine, and 20 ug/ml creatine kinase, in addition to sSDNA, dsDNA, and proteins at the concentra- tions given in the figure legends. For the reactions in Fig. 8, mixtures (3 yl) Were incubated at 37-C for 30 minutes. For the reactions ~n ~ i ~ . 9 , uvsx

- XhoI termini by using the Xlenow fragment of E. coli polymerase I (New

protein was incubated for 5 minutes at 3 7 T with all components except 32 protein, ATP, and dsDNA. 32 protein and ATP Were then added and the incuba- tion continued for 5 minutes. dsDNA was added, and the complete mixture ( 3

of stop solution (23% glycerol, 75 mM EDTA, 0.06% bromophenol blue, and 5.6% u1) then incubated for 30 minutes. Reactions were stopped by adding 1.2 "1

proteins from DNA. The DNA products Were Separated by electrophoresis on a

SDS): the incubation was continued for 5 minutes at 37°C to dissociate

1.2% agarose gel (7 cm x 10 cm) (mini slab gel) for 3 hours at 40 V and

with Kodak XR-5 film and a DuPOnt Cronex Plus-X intensifying screen (Fig. 9). either stained with ethidium bromide (Fig. 8) or dried and autoradiographed

acrylamide, SDS gels was performed as described (31). Other Pethods - Electrophoresis of proteins through discmtimous poly-

with or without plasmids or infected with T4D (at moi of 5). as described Extracts for 8-glucoayltransferase assays were prepared from OH1 cells,

above (see '"Fractionation of Extracts from & and & Cells by Double- Stranded DNA Cellulose Chromatography") except that cells were sonicated in 0.05 M potassium phosphate buffer, pH 7.4. Cleared lysates were then ob- tained after centrifugation at 10.000 x 4. Aliquot$ of these lysates or of the 0.5 M NaCl eluates from dsDNA cellulose columns (see above) were assayed for E-glucosyltransferase activity essentially as described (44) in a re-

bacteriophage T2 DNA (nucleotide equivalents), 25 m MgC12, and 50 UM uridine action mixture 114 ull containing 100 mM potassium phosphate, pH 7.8. 60 pM

diphosphate glucose Igl~cose-~~CI (0.026 Ci/mmol). After reaction mixtures Were incubated at 30'C for 15 minutes, transfer of glncose to DNA was deter- mined by acid-insoluble radioactivity (45).