Gene, 104 (1991) 103-106 0

1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved

103

0378-l 119/91/SO3.50

05058

Sequence of RAD54, a Saccharomyces (Double-strand

break repair;

cerevisiae gene involved in recombination and repair

nucleotide-binding

site; DNA damage

induction;

yeast; recombinant

DNA)

Herschel1 S. Emery a*, David Schildb, David E. Kellogg” and Robert K. Mortimer”vb u Department ofMolecular and Cell Biology, Universityof Calgornia, Berkeley, CA 94720 (U.S.A.), and ’ Divisionof Cell andMolecular Biology, Lawrence Berkeley Laboratory, Berkeley, CA 94720 (U.S.A.) Tel. (415)643-8877 Received by J. Marmur: 9 April 1991 Revised/Accepted: 1 May/2 May 1991 Received at publishers: 15 May 1991

SUMMARY

The complete nucleotide sequence of the RAD.54 gene of the yeast Saccharomyces cerevisiae has been determined. The sequenced region contains an open reading frame of 2694 bp, and the predicted RAD54 protein has a potential nucleotidebinding site and possible nuclear targeting sequences. Northern analysis reveals a transcript of approx. 3.0 kb which is induced following x-ray irradiation.

INTRODUCTION

The RAD54 gene of the yeast S. cerevisiae is involved in both DNA repair and mitotic recombination. Mutations in this gene cause extreme x-ray sensitivity (Game and Mortimer, 1974) the inability to repair double-strand DNA

breaks (Budd and Mortimer, 1982) and decreased spontaneous and induced mitotic recombination (Saeki et al., 198 1). This gene was cloned by complementation of a rad.54 mutation and the location of this gene on the cloned fragment was determined by subcloning experiments (Calderon et al., 1983). Regulation studies on RAD.54, using IacZ

RAD54

548 RNA (-1.4 kb)

RNA

(-3.0 kb)

1 kb

I

I Fig. 1. Transcription restriction

map of the RAD54 region.

The gene encoding

sites were used to isolate probes which hybridized

(BglII sites) (see Fig. 2). The solid line represents on the RAD54 transcripts

represents

Correspondence to: Dr. R.K. Mortimer, Cell Biology, 102 Dormer Laboratory,

of its transcription,

and the hatched

Department University

Fax (415)642-8589. Department of Biology,

Richmond, VA, 23173 (U.S.A.) Tel. (804)289-8240.

(54B) has not been characterized. (BamHI

boxes are the two transcripts boxes and dashed

The underlined

sites) or both the RAD54 and 54B transcripts encoded

by this insert, the arrow

lines are part of the YEpl3

vector. The order

has not been determined.

of Molecular

of California,

and

Berkeley,

Abbreviations:

aa, amino

1000 bp; nt, nucleotide(s);

acid(s);

University

of Richmond,

bp, base pair(s);

ORF, open reading

RNA; S., Saccharomyces; tRNA, point(s).

CA 94720 (U.S.A.) Tel. (415)643-8877; * Current address:

1.4-kb transcript

to the RAD54 transcript

the cloned yeast insert, the blackened

the direction

of the BglII and Hind111 sites in parentheses

the approx.

specifically

transfer

kb, kilobase

frame; rRNA,

or

ribosomal

RNA; tsp, transcription

start

kb

234567A

Fig. 2. Northern-blot

analysis

RAD54. All lanes contain

3.4 -3.0

2 and 4) and irradiated

of RAD54 region and x-ray induction

15 pg of total RNA from unirradiated (lanes 5-8) diploid wild-type

X2180, except lane 3 which contained cells. Lane I was probed with

fragments

Lanes 5-8 contain

1.8

containing

60 and 90 min, respectively,

RAD52,

RAD54

after treatment

lane 3 was loaded

-1.4

cells) as lane 4, the induction

only RAD54, lane 2

and

54B

sequences.

with 20 krads of x-ray. Since

with three times as much

to be between

18s rRNAs

containing

and the rest of the lanes were probed

RNA from cells which were allowed to grow 15, 30,

-1.6

appears

S. cerevisiue strain

45 pg of RNA from unirradiated

with a fragment

was probed with a RAD52 fragment

of

(lanes 1,

RNA (from unirradiated

ratio of the RAD.54 transcript

five- and ten fold. The mobilities

were determined

from the ethidium

(lanes 5-8)

of the 25s and

bromide-stained

gel and

used as size standards

a

V

I-

+

A

v

v

a

+

w

w

nt 270-

nt

260-

310281 271 250 234-

230

220

-

23456

Fig. 3. S I mapping gel. Lanes:

of

fspof RAD54. (Panel A) Autoradiogram

1, size standards:

and treated

with S 1; 3, Sl protection with its complementary

1977). (Panel B) DNA sequencing lane 3); 2-6, DNA sequencing

experiment:

denatured

strand,

although

reaction

end-labeled

samples

conditions

Bg

denatured

for specificity

of the labeled probe protected

5% polyacrylamide/8.3

,

of end-labeled

1$X174 RF DNA- Hue111 cut; 2, control

lower two bands in lane 3 are fragments by reannealing

ORF

_

Bg B

DNA run on a low-resolution

ofprotection:

probe was mixed with unlabeled by RAD.54 mRNA,

probe.

5”/b polyacrylamide/8.3

end-labeled

total yeast RNA prior to Sl digestion.

hybrids

fragments

M urea

probe mixed with E. coli tRNA

while the top band is a full-length

were used to favor RNA-DNA

M urea gel. Lanes: 1, Sl-protected

run on the full-length

denatured

over DNA-DNA

of end-labeled

probe, probably hybrids

The

protected

(Berk and Sharp,

probe (same sample as in panel A,

All lanes are from the same gel and adjacent,

but because

the bands

in

lane 1 are faint, this lane has been photographically overexposed. A gap was left between lanes 1 and 2 to decrease cross contamination with the sequencing reaction samples. The arrows on the right margin represent tsp.At the bottom of this figure is a diagram of the yeast chromosomal region near the start of the RAD54 gene, the two (actually major classes of transcripts.

The thick line represents

specify the “P label. B, BarnHI,

Bg, Bg/II.

four, but three are very close together)

tsp(5’mRNA;

vertical

lines; see Fig. 4), and the inferred

part of the RAD54 ORF and the arrow above it shows the direction

of transcription.

two

Stars (dots)

105 fusions,

have shown that it is transcriptionally

induced

by

EXPERIMENTAL

AND

DISCUSSION

DNA damage (Cole et al., 1987) and by entry into meiosis (Cole et al., 1989). Approximately 300 bp of the 5’ regulatory region has been sequenced independently and a 29-bp region has been identified which contains the sequence responsible for damage induction (Cole and Mortimer, 1989). The 5’ region also contains a copy of a consensus sequence which is involved in the cell-cycle regulation of a number of yeast genes (McIntosh et al., 1991), but cell cycle regulation of RAD54 has not been tested. To

(a) Transcriptional analysis The Northern analysis reported here shows that the original, approx. 6.2-kb genomic clone containing RAD54 encodes two transcripts of approx. 1.4 and 3.0 kb (Figs. 1 and 2). Both the original subcloning experiments (Calderon et al., 1983) and subsequent gene disruption experiments (D.S. and R.K.M., unpublished) demonstrate that the longer transcript is that of the RAD54 gene. The size of this

further characterize RAD54, analyzed its transcription.

transcript discussed

we sequenced

this gene and

also tits well with the size of the RAD54 ORF below, and suggests that, like most genes of

AAAGCTTATGTATCAAAAATTTAACATCTTGAAAATCTTG-TACAC~GTGGTGC~GATGTGTCACGTTCTGGACCTGAGTGGTGCCATGTATGCTATTT~CATGC~GGGG~GACCCTTC CGCCTTACTGCAATAATAGTATTTTACGCGTTACCC~TATAGC~GTTTCGCGC~T~C~TTAC~C~G TAACTGAAG~GAAGGCC-CTCTTCTCACTTGACGTACTTTTCTCTTTCTTCACTAAAGCTGCTACGAAAGTATAG

120 240 360

AAAAAAAAGGAAATAATAGAAGATC AAAAATCAAACGCT

MARRRLPDRPPNGIGAGERPRLVP

24

CAGAACTTAGCTCTATTTCAGGTACCATATATATTTCCTTAT~CTGATGGC~GACGCAGATTACCAGACAGACCACC~TGG~TAGGAGCCGGTG~CGGCCGAGACTGGTACCT SVNRLTKPFRVPY&NTHIPP AAGRIATGSDNIV N V Q D _BPI AGGCCTATTAACGTACAAGACTCGGTGAACCGACTAACGACT~CG~CCGTTCAGGGTCCCGTAC~G~CACGCACATCCCGCCCGCTGCTGGTAG~TCGCCACCGGGTCTGAT~TATCGTA GGRSLRKRSATVCYSGLDINADEAEYNSQDISFSQLTKRR GGAGGAAGGAGCTTGAGGAAAAGATCAGCGATCAGCGACTGTATGTTATTCCGGCTTGGATAT~TGCGGACG~GCAGAGTAC~CAGCC~GACAT~GTTTTTCTCAGTTGACT~CGACGG KDALSAQRLAKDP TRLSHI QYTLRRSFTVPIKGYVQRHSL AAGGATGCTCTTAGTGCTCAGGTTGGCCAAGGATCCGATCCGAC~GACTGAGTCATATCCAGTACACTTTGAG~GATCTTTCACTGTGCC~TC~GGGATATGTAC~GACACAGTCTG PLTLGMKKKITP EPRPLHDP TDEFAI V L Y D P SVDGEMIVH CCATTAACGTTAGGAATGAAARRAAAAA TTACTCCAGAACCTCGACCTCTACATGACCCTACAGACGATGATTGTCCAT DTSMDNKEEESKKMIKSTQE K D N I N K E KNSQEERP GACACATCTATGGATAACAAGG~G~G~TC~G-TGATC-GCACACAGG-GGAT~TATT~C~GG-G~TAGCCAGG~G~GACCCACAC~G~TAGGA RHPALMTNGVRNKPLRELLGD SENSAENKKKFASVPVVID CGCCATCCAGCTTTGATGACAAATGGTGGTGTGAG~C~CCGTTACGTGAGCTACTCGGTGATTCGG-CTCTGCGG-C~G~G~TTTGCTAGTGTCCCTGTGGTTATTGAT PKLAKI L R P H Q VEGVRFLYRCVTGLVMKD YLEAEAFNTSS CC-CTGGCCAAGATTTTAAGACCCCCATC~GTCG~GGGGTGAGATTCTTATACCGTTGTGTCACAGGGCTTGTCATG~GATTACTTAG~GCTGAGGCGTTC~CACTTC~GC E D P LKSDEKALTESQKTEQNNRGAYGC IMADEMGL

480 64

T

Q

R

GKT

I

G

L

Q

GRAGATCCCTTRAAGAGCGACGAGAAGGCACTTACTTACTG~TCTC~GACTG~C-C~TAGAGGGGCCTACGGCTGCATCATGGCCGATG~TGGGGCTGGGT~GACATTGCAG CIALMWTLLRQGPQGKRLI D K C I IVCPSSLVNNWANELIK TGTATAGCACTTATGTGGACATTATTAAGACAGGGGCCCGCAGGGT-GACTTATCGAC~TGTATCATTGTTTGTCCATCTTCTTTAGTC~C~CTGGGCT~TG~TTGATWLGPNTLTP LAVDGKKSSMGGGNTTVSQAIHAWAQAQGRN TGGTTGGGTCCCAATACTCTRACTCCGTTAGCGTTAGCGGTTGATGGGW TCTTCAATGGGTGGTGGTAATACRACAGTTTCGCRAGCTAT IVKPVLII SYETLRRNVDQLKNCNVG LMLADEGHRLKNGD ATTGTTAAGCCCGTTTTRATCATATCGTATG~CATTACGTCG~TGTTGATC~TTG~CTGC~TGTGGGTTT~TGCTGGCAGATG~GGGCACCGTTTG~CGGTGAC SLTFTALDSI SCPRRVILSGTP I Q N D L SEYFALLSF S N TCTTTAACATTCACAGCTTTGGATAGCATCAGTTGTCCTAG~GAGTTATATTATCTGGTACACCCATTC-CGATCTTTCCG~TATTTTGCTCTACTGAGTTTTTCG~TCCTGGC LLGSRAEFRKNFENP ILRGRDADATDKEI TKGEAQ L Q K TTATTAGGTTCCAGAGCAGAATTTTAGG~TTTTGAG~TCC~TTTTGCGAGGGCGTGATGCTGATGCTACTGAT~G~TCAC~GGGCGAGGCGC~TTACAG~GCTATCT T IVSKFII RRTNDI LAKYLP CKYEHVIFVNLKP L Q N E L ACCATTGTCTCCAAATTTATTATCCGTCGTACT~CGATATTTTGGCC~TATTTGCCTTGC~GTATGAGCACGTTATTTTCGT~CTTG~GCCGTTGCAG~CGAGCTCTAC~T K L IKSREVKKVVKGVGGSQP LRAIGI LKKLCNHPNLLNFE AAATTAATTAAGTCAAGAGAGTGAAAAAA GTAGTAAAAGGTGTTGGTGGGTCTCAGCCCCTGAGGGCTATTGGTATTTT~G~GCTATGT~TCACCCT~TCTCTTG~TTTTG~ D E F D D E D D LELPDDYNMPGSKARDVQTKY SAKFSILERFL GATGRATTTGACGATGAAGATGATTTGGAACTACCTACCTGACGATTAT~TATGCCGGGTTC-GCCAGGGACGTAC~C-TATTCAGCT~TTTTCCATTTTGG~GATTTCTC H K I KTESDDKIVLISNYTQTLD LIEKMCRYKHYS AVRLDG CATAAGATAAAGACCGA;LTCCGATGATAAGATTGTTGTTTTGATCTCC~CTATACCC~CCTTGGACCTCATTG~ TGTGTAGATATAAGCACTACAGTGCTGTACGGTTGGATGGT TMSINKRQKLVDRFNDPEGQEF IFLLSS KAGGCG INLIGA ACAATGTCAATTAACAAAAGACAAAAATTGGTGGTGGATAGGTTC~CGATCCTG~GGCC~GAGTTCATCTTCCTCTT~GTTCT~GCAGGTGGGTGTGGTATC~TTTGATTGGTGCA N R L ILMDPDWNPAADQQALARVWRD GQKKDCFI Y R F I S AATCGATTAATTTTGATGGATCCGGATTGGAATCCTGCTGCTGCT~TC~C~GCTTTGGCACGTGTTTGGAGAGACGGTC~ GGATTGTTTCATTTACAGATTCATATCGACTGGT T I EEKIFQRQSMKMSLS SCVVDAKEDVERLFSSDNLRQLF ACCATAGAGGW TCTTTCAAAGACAATCTATGAAAATGAGTTTGAGTTTGAGTTCATGCGTGGTCGATGCG~G~GACGTTG~GGTTGTTTAGTTCTGAC~TTT~GACAGTTGTTT QKNENTICETHETYHCKRCNAQGKQLKP APAMLYGDATTW CAAAAAAATGAGAATACGATATGTGAAACACACACATTTAAAGAGAGCCCCTGCAATGTTATATGGTGATGCGACAACTTGG NHLNHDALEKTNDHLLKNEHHYNDI SFAFQYISH AATCATTTGAACCATGACGCATTGG AAAAAACAAATGATCATCTACTAAAAAA CGAGCATCATTACAATGATATCAGTTTTGCATTTCAATATATTTCACATTGATCTCTTACATACATG TACTTAT-CAAAAACAGTAGAATTCGATCGATCGTCGGGGGGTCATTACATATTTATTTT~TGATGGGCATGGTTATATATATTTAGACGT~T~C~TTG-TTGGGGCC AAATTGGTAAATATCTCTATGTATTTCAATC AAAAAAGTATAATCGTATAACTTAAGGGTGTTGAGCGATGTCATATTATACTTGTGCTAGCTTTTAAATTTTTCATTATCTT CAGTTTTATCGTGGTRAAGTGCCAGCCGAAACTGCC AAAAAAGATTATGGCAAG-TCTCACGCTGTCAGGGAAATAAAAA TCTGATACAGTTTCACACTCTCAGAAGGATTAACTTT AACTATTACCTAACACCRATATCTGAAGAGAG~TGGATG~TTCTTGCC~GCAGGTTCAC~GCCGTTACATTTGCTAT-TCCGG~TATCGATAGCATCCACATATGCGCT~ GACAATAACGAAC

Fig. 4. Nucleotide

sequence

sequences were determined in the ORF (nt 1072-1080

of the RAD54 gene of S. cerevisiae. The Maxam independently for both strands. The unavailability and 2654-2660). The final 40 nt of 3’-untranslated

gene are underlined (nt at 250,281,282 and 283). Two potential nuclear-targeting (aa 340-342) are underlined. The GenBank accession No. is M63232.

and Gilbert

(1980) chemical

sequencing

technique

P

G

L

S

Y

N

T

G

600 104 720 144 840 184 960 224 1080 264 1200 304 1320 344 1440 384 1560 424 1680 464 1800 504 1920 544 2040 584 2160 624 2280 664 2400 704 2520 744 2640 704 2760 824 2880 864 3000 898 3120 3240 3360 3480 3600

was used, and the

of suitable restriction sites prevented confirmation of two short sequences sequence were determined for one strand only. The multiple cspfor this sequences

(aa 21-25 and 41-45) and an ATP-binding

consensus

sequence

S. cerevisiae, this gene lacks introns. The second transcript present on our genomic clone has arbitrarily been called 54B. Our analysis also confirms that the RAD.54 transcript is induced by x-ray treatment, but that neither the 54B nor the RAD52 transcripts is induced. Previously, the lack of DNA-damage induction of &ID52 had only been shown with 1ac.Z fusion studies (Cole et al., 1987).

(b) Sl mapping of transcription start points (tsp) Sl mapping (Berk and Sharp, 1977) was performed

to

determine the tsp for RAD54 transcription. A suitable hybridization probe, 5’ end-labeled (using polynucleotide kinase and [ y-32P]ATP) at a HinfI site approx. 100 bp into the ORF, was prepared. The probe extends from this HinfI site to a site approx. 400 bp upstream from the presumed initiator AUG. The labeled probe was then hybridized to total RNA prepared from unirradiated diploid cells. Hybrids thus formed were then treated with S 1 nuclease to remove the unprotected portion of the probe upstream from the tsp, The electrophoretic pattern of protected fragments (Fig. 3A, lane 3) shows two prominent bands, suggesting tsp approx. 250 and 220 nt upstream from the IIinfI site. To refine the location of these 5’ ends of the transcript, the Sl-protected fragments were sized in a high-resolution DNA sequencing gel alongside nt sequence ladders for the full-length probe. The pattern of protected fragments (Fig. 3B, lane l), while faint, is fully consistent with the earlier pattern, showing a band 253 nt in length and a triplet of bands 220, 221 and 222 nt long.

transcript seen by Northern analysis. The RAD54 transcript is induced by DNA damage, and multiple t.sp are seen for this gene.

ACKNOWLEDGEMENTS

This

work

was

supported

Glassner

and Gail Christie

from

NIH

for useful discussions.

REFERENCES Berk, A.J. and Sharp, mRNA

and mapping

P.A.: Sizing

of early adenovirus

of S 1 endonuclease

by gel electrophoresis

digested

hybrids.

Cell 12 (1977) 721-732. Budd,

M. and Mortimer,

temperature

cerevisiue. Mutation Calderon,

of double-strand mutant

breaks

in a

of ~~~&~u~~~~~~e.~

Res. 103 (1982) 19-24. CR.

ofyeast

complement

Repair

radiatjon-sensitive

I.L., Contopoulou,

characterization that

R.K.:

conditional

the

rud55-3. Curr. Genet.

and Mortimer,

R.K.: Isolation

DNA repair genes, II. Isolation radSO-1, rarlsl-l,

mutations

and

of plasmids rad54-3,

and

7 (1983) 93-100.

Cole, G.M. and Mortimer,

R.K.: Failure

to induce a DNA repair gene.

R.4D54, in Saccharomyces cerevbiae does not affect DNA repair recombination phenotypes. Cole, G.M., Schild, D., Lovett,

RAD54- and RADSZ-la& fusions sponse

to DNA damage.

Mol. Cell. Biol. 7 (1987) 1078-1084. R.K.: Two DNA repair and recom-

genes in Saccharomyces cerevisiae, MD52

induced

during

meiosis.

J.C. and Mortimer,

tosidase Landschulz,

R.K.: A genetic

L. and Herskowitz,

W.H.,

binding

proteins.

Maxam,

P.F.

structure

Science

A.M. and Gilbert, E.V., Atkinson,

tion of a short stage-dependent

off?. cob &galac-

I.: Targeting

and

McKnight,

common

S.L.: The leucine

to a new class of DNA

240 (1988) 1759-1764.

W.: Sequencing

specific chemica1 cleavage. McIntosh,

mu-

in yeast. Cell 36 (1984) 1057-1065.

Johnson,

a hypothetic~

study of x-ray sensitive

Res. 24 (1974) 281-292.

to the nucleus

zipper:

and RAD.54, are

Mol. Cell. Biol. 9 (1989) 3101-3104.

tants in yeast. Mutation Hall, M.N., Hereford,

of

in Saccharomyces cerevisiae in re-

bination Game,

or

Mol. Cell. Bid. 9 (1989) 3314-3322. S.T. and Mortimer, R.K.: Regulation

Methods

end-labeled Enzymol.

DNA with base-

65 (1980) 499-560.

T., Storms, R.K. and Smith, M.: Characteriza-

c&-acting

DNA

transcription

sequence

which

conveys

cell cycle

in Saccharomvces cerevisiae. Mol. Cell.

Biol. 11 (1991) 329-337. Naumovski, repair

L. and Friedberg, functions

mutagenesis.

E.C.: Analysis

ofthe essential

and excision

of the RAD3 gene of Saccharomyces cerevisiae by

Mol. Cell. Biol. 6 (1986) 1218-1227.

Reynolds, P., Weber, S. and Prakash, L.: The nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae: a potential adenine nucleotide binding amino acid sequence

(d) Conclusions The nt sequence of the yeast RAD-54 gene reveals an ORF or 2694 bp, which is consistent with the approx. 3.0-kb

grants

(GM30990 and NRSA training grant ES07075), and from the Off&e of Health and Environmental Research, Office of Energy Research, U.S. Department of Energy, under Contract DE-AC03-76SF00098. We wish to thank Brian

Cole, G.M., Schild, D. and Mortimer,

(c) The nt sequence analysis Here, we also report the nt sequence of the entire RAD54 gene, which encodes one large ORF of 2694 bp (Fig. 4). The predicted aa sequence of the RAD54 protein contains a sequence (underlined GKT aa) similar to the ATP-binding consensus sequence found in many proteins (Naumovski and Friedberg, 1986). Two potential nuclear-targeting sequences are also present (underlined RLVPR and RVPYK aa) (Hall et al., 1984). A potential leucine zipper is encoded by nt 1696-1761 (Landschulz et al., 1988), and a 5-aa region (ARRRL) has been found to be shared by the RAD.54 and RAD6 proteins, at aa 2-6 and 5-9, respectively (Reynolds et al., 1985). The RAD54 protein is quite basic, particularly the N-terminal end, which is also very proline-rich. No significant homologies have been found between the inferred RAD54 aa sequence and other proteins and inferred proteins present in recent data bases.

by

terminal

and a non-essential

acidic carboxyi

region. Mol. Cell. Biol. 7 (1985) 1012-1020.

Saeki, T., Machida, I. and Nakai, S.: Genetic control of diploid recovery after y-irradiation in the yeast Saccharomyces cevevisiae. Mutation Res. 73 (1981) 251-265.