Isolation and characterization of the Schizosaccharomyces pombe rad3 gene, involved in the DNA damage and DNA synthesis checkpoints

Gene, 119 (1992) 83-89 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

83

0378-l 119/92/$05.00

06660

Isolation and characterization of the Schizosaccharomyces pombe gene, involved in the DNA damage and DNA synthesis checkpoints (Recombinant

DNA;

radiation

resistance;

DNA repair;

rad3

cell cycle control)

Brent L. Seaton *, Jennifer Yucel, Per Sunnerhagen

* and Suresh Subramani

Departmentof Biology, University of California, San Diego La Jolla, CA 92093-0322, USA Received

by R.M. Harshey:

30 May 1992; Revised/Accepted:

10 May/l

1 May 1992; Received at publishers:

11 June 1992

SUMMARY

We have cloned the Schizosaccharomycespombe rad3 gene which is involved in G2 arrest following DNA damage, and in the dependence of mitosis on the completion of DNA replication. The gene was cloned by complementation of the sensitivity to UV light and y rays of the rad3-136 mutant with an Sz. pombe genomic library. Sublocalization of the complementing activity and sequencing of the clone identified an intronless 3210-bp open reading frame capable of encoding a 1070-amino acid protein with an M, of 121974. The rad3 gene is a new gene with no homologs in existing sequence databases. The gene is poorly expressed, with a codon bias index of -0.01. A disruption mutant affecting the coding region was only slightly more sensitive to UV light than the original radjl-136 mutant. The rad3 gene was mapped to Not1 fragment C on chromosome II.

INTRODUCTION

Living organisms are continually exposed to a variety of mutagens many of which are demonstrably toxic to cells. The survival of the organism, and the propagation of the species, are therefore critically dependent upon cellular

Correspondence to: Dr. S. Subramani, Jolla, CA 92093-0322, * Present addresses: California

USA. (B.L.S.)

Department

Tel. (619) 534-3092; Department

College of Medicine,

University

of Biology, UCSD,

La

Fax (619) 534-0053.

of Physiology

and Biophysics,

of California

at Irvine, Irvine,

CA 92717, USA. Tel. (714) 856-6541; (P.S.) Departments of Molecular Biology and Medical Biochemistry, University of Goteborg, P.O. Box 33031, S-400 33 Goteborg, Abbreviations: codon

Sweden.

aa, amino acid(s);

bias index; ds, double

dium; kb, kilobase

Tel. (46-31) 85 30 00.

Ap, ampicillin;

strand(ed);

EMM,

bp, base pair(s); essential

or 1000 bp; MM, minimal medium

Nm, neomycin; npt, Nm phosphotransferase-encoding otide(s); ORF, open reading frame; PFGE, pulsed-field

minimal

CBI, me-

(see section a); gene; nt, nuclegel electrophore-

sis; S., Saccharomyces; Sz., Schizosaccharomyces; ss, single strand(ed); UV, ultraviolet light; UVR, UV resistance; YPD, 1% yeast extract/22 peptone/2% dextrose medium.

mechanisms which reverse the damage inflicted by such agents. Until recently, the budding yeast Saccharomyces cerevisiae served as the predominant paradigm for the study of these DNA repair processes in eukaryotes. However, in the last few years the fission yeast, Sz. pombe, has emerged as an attractive complementary organism for the investigation of DNA repair and recombination. Sz. pombe is inherently very radiation-resistant (Subramani, 1991). Though it has approximately the same cell size and genomic complexity as S. cerevisiae, it can withstand approximately tenfold higher doses of UV light or ionizing radiation (Phipps et al, 1985). Therefore it must possess efficient means for repair of DNA damage. Numerous radiation-sensitive (rad) Sz. pombe mutants exhibiting increased sensitivity to UV light and/or ionizing radiation have been isolated and classified into 21 complementation groups based on allelism tests (Nasim and Smith, 1975; Lieberman et al., 1989; Schmidt et al, 1989). The rad3-136 mutant is sensitive to UV light (Nasim and Smith, 1975), and exhibits a reduced frequency of UVinduced forward mutation as compared to wild-type cells

84 (Gentner et al., 1978). Recently we have found that the md3 mutant is deficient in G2 arrest after DNA damage by y-irradiation and in the coupling of mitosis to DNA replication (Jimenez et al., 1992). In this paper we describe the isolation and characterization of the rud3 gene by complementation of the Sz. pombe rad3-136 mutant with an Sz. pombe genomic library.

and the resulting plasmids were ligated into pFL20 which had been linearized with BamHI. Individual plasmids were again introduced into Sz. pombe h - rad3-136 Ural and Ura + transformants were tested for complementing activity by spot assays. Two plasmids, pSub1.41 (Fig. 1A) and pSub2.6 (data not shown) with insert sizes of 9.8 and 4.0

AlO_. RESULTS

AND DISCUSSION

(a) Cloning of the rud3 gene by genetic complementation Sz. pombe h _ rud3-136 uru4 (a gift from A. Nasim) was transformed with a library of genomic Sz. pombe DNA (a gift from John Carbon; Elliot et al., 1986). This library contains Sz. pombe DNA partially digested with Sau3A inserted into the BamHI site of pFL20 (Losson and Lacroute, 1983), which carries the S. cerevisiae WRA3 gene, capable of complementing the Sz. pombe ura4 mutation. Approximately 1.2 x lo4 Ura+ tr~sformants were obtained when 6 x IO8 cells were transformed with 60 pg of library DNA (Beach et al., 1982) The transformants were washed off the plates and diluted to a density of 4 x lo6 cells/ml in MM (0.67% yeast nitrogen base without aa/2% glucose and appropriate supplements/75 pg per ml of adenine/40 pg per ml each of histidine, leucine, and uracil). An aliquot of this suspension was then irradiated at 120 or 180 J/m* with agitation, and thereafter plated onto MM plates lacking uracil. After two days of growth, random survivors were picked and cultured overnight in liquid MM. The rem~ning colonies on the plates were pooled and again irradiated at 120 or 180 J/m2. As described above, after two days of growth random survivors were picked into liquid MM. For each colony sample, a small volume of the survivor cultures was used for spot assays. Out of 105 survivors retested for complementation of the rad3-136 mutant as judged by the spot assay, 37 appeared Rad’, as defined by the ability to survive a UV dose of 180 J/m2. Colonies e~ibiting enhanced resistance to UV irradiation were tested for stability as described by Sunnerhagen et al. (1990). In each case (37/37), the clones had become Ura- and UV-sensitive. This suggests that the complementing activity resided on an extrachromosomal plasmid which was lost under conditions of nonselective growth. Plasmid DNA was isolated from the UV-resistant clones and used to transform E. coli DH5a to Ap resistance. Plasmids were individually introduced back into Sz. pombe h - rud3-136 ura4 and complementing activity was determined by spot assays. Out of 18 individual DNAs tested the smallest plasmid giving full complementing activity contained an insert of approximately 15 kb. This plasmid was subjected to partial Suu3A digestion

0

so

46

UV

0

20

40

135

180

225

Dose (J/a&

60

80

100

120

140

Dose (krad)

Fig. 1. Complementation mutant

by plasmid

rich YPD medium.

of the UV- and y-ray sensitivity of the r&3-136

pSubl.41.

Yeast cells were grown

Selective growth

nonselectively

in

of cells was in MM. Cells were in-

cubated at 30°C for vegetative growth. A 15-W Sylvania G15T8 germicidal lamp emitting primariiy 254~nm light was used for UV i~adiation. The dose rate was 3 W/m2 as measured

by a Blak Ray 5225 short-wave

UV meter, For irradiation

of Sz. pombe with y-rays, cells were suspended

in MM at a concentration

of 4 x lo6 cells/ml and irradiated

with a r3’Cs

source for various times at a dose rate of 1 krad/min. Quantitative measurements were made by growing the Sz. pombe strain overnight in liquid, selective or nonselective

medium

tion of 4 x lo6 cells/ml.

Cells in suspension

and then diluting cells to a concentrawere irradiated

at various

doses while being agitated and then plated on selective or nonselective solid media at various dilutions. Each data point is the average of colonies observed in at least two experiments. Squares, 972 h ; triangles, hr&3-136 urn$; circles, hm r&3-136 Ural transformed with pSubl.41. (A) Survival of the strains after UV-i~ad~atjon. (B) Survival after ~~-irradjation. Tr~sfo~at~on of the h- rad3-136 ura4 strain with the pFL20 vector did not affect resistance

to UV- or y-radiation

(data not shown).

85 sition 3270 completely abolished the ability of pSub 1.4 1 to complement the rad3-136 mutation.

kb, respectively, complemented the UV-sensitive phenotype of the r&3-136 mutant. Cells containing the plasmid pSub2.6 exhibited unstable growth. The plasmid pSubl.41 also complemented the sensiti~ty of the rad3-136 mutant

(c) Plasmids pSub2.6 and pSubl.41 contain the rad3 gene In order to verify that the subclones did indeed contain the rad3 gene and not a suppressor of the rad3-136 muta-

to y-irradiation (Fig. 1B). Restriction maps of pSub2.6 and pSubl.41 are shown in Fig. 2A. The inserts contain an overlapping region of 3.7 kb. The plasmids were colinear to each other, as well as to

tion, a genetic approach was taken. Plasmid pR3Hl.OLEU2 (Fig. 4) was linearized

the Sz. pombe genome, as determined by Southern blot analysis (Fig. 3). Because of the growth instability of pSub2.6-tr~sfo~ed SZ. pombe cells, and the fact that pSub2.6 and pSubl.41 clones were colinear within their overlapping region, the larger pSub 1.4 1 subclone was used for functional analysis, and both pSub2.6 and pSubl.41 clones were used for sequence analysis.

(b) Functional localization of the rud3 gene within pSubl.41 Plasmids resulting from the insertions of the npt gene into different portions of the rad3 gene were individually introduced into Sz. pombe h- rad3-136 Ural and Ura+ transformants were assayed for their resistance to UV irradiation. As shown in Fig. 2B, insertions into sites ranging from the MluI site at nt position 259 to the NheI site at nt poM Sn I i I I

H I I

I 1

A.

Sn f I

I 2

H I I

XNh I 1 ,I

I

3

4kb

pNhbneo

Fig. 2. Restriction and pSubl.41

map, sublocalization

(lower) containing

to define functional

of complementing

activity and sequencing

the rud3 gene. Abbreviations:

regions of pSubl.41.

In order to determine

was inserted

(indicated

by the triangles)

of pSV2Neo

(Southern

and Berg, 1982), which contains

pSubl.41

was linearized

with various

restriction

strategy.

restriction

(A) Restriction

M, M&I; N, NheI; Sn, SnuBI;

the region of pSubl.41

fragment Plasmid

into various

H, HindHI;

fragment

responsible

-

maps of overlapping

clones pSub2.6

X, Xhol. (B) Position

for the r&3-complementing

sites within the area of overlap with pSub2.6

the Tn5 npt gene, was isolated

enzymes

at the

unique ClaI site at position 2307 of the rad3 sequence, and used to transform Sz. pombe h- his3 leul-32 ura4D18 (a gift from P. Russell) to leucine prototrophy. Southern blot analysis (data not shown) verified that plasmid pR3H 1.OLEU2 had integrated at the Sz. pombe locus from which the complementing sequence had been derived. Since the 1057-bp Hind111 fragment is internal to the putative rad3 gene, integration of pR3Hl.OLEU2 into the above Rad’ strain created a UV-sensitive mutant. Two genetic crosses were done to show that pR3Hl. 0LEU2 integrated at the rad3 locus. Crosses were done on EMM plates incubated at 25 ‘C as described by Gutz et al. (1974). Asci were treated with fi-glucuronidase (Sigma Chemical Co.) at 37°C overnight before plating spores on nonselective plates.

(upper)

of npt insertions

activity,

used

a foreign DNA

(part B). The 1.3-kb HindIII-NruI

and the ends were filled in with T4 DNA polymerase.

and the ends were made blunt with T4 DNA polymerase.

was ligated into pSubl.41 at the respective locations, and the ligation mixtures were used to transform the position of insertion of the npt fragments into pSubl.41 in relation to its restriction map above.

The 1.3-kb npt fragment

E. coli to Ap and Nm resistance. Triangles show npt fragments were inserted into the MluI, SnuBI,

XhoI and NheI sites. Plasmids containing the insertions were introduced into the h- rad3-136 ura4 strain and the phenotype scored after UV irradiation is shown at the right. Lack of restoration of UVK is indicated by a minus sign. For qualitative measurements of UVR, Sz. pombe colonies were grown overnight

under selective or nonselective

irradiated

at various

box and corresponds Fig. 5. Arrows

doses.

Survival

conditions

was recorded

to the scale of restriction

represent

portions

in liquid medium.

map A. Construction

of the nt sequence

The cell suspensions

after 48 h of growth

were deposited

at 30°C.

(C) Sequencing

of sequencing

substrates

derived from specific subclones.

as 5-~1 droplets

onto solid medium plates and

of r&3 gene. The 3210-bp

and sequencing

The sequence

procedure

derived from pSub2.6

ORF is shown as an open

are described

in the legend to

begins at bp 129.

123456789 2767 bp

1711

1057-bp Hind111 fragment of pSub2.6

t HindIII-digested pLEU2

Y

Ligate to create pR3Hl.OLEU2 Digest at unique ClaI site Transform ura4 leul his3 hSelect for La+

I

I 1

rad3 locus

4100 bp ura4 leul his3 h _ W-resistant

I

2767

1

h-7-_ rad3

-

LEUZ

1711

4100 bp i-ad3 -

rad3 ura4 his3 W-sensitive

Fig. 3. Plasmids colinear.

pSub2.6,

DNA fragments

rud3 subclone

plasmid

an agarose

gel, transferred fragment

genomic

DNA.

obtained

to nitrocellulose,

of pSub2.6

Lanes

plasmid

DNA.

XbaI which has sites at nt positions fragment.

and probed

on

Lanes

plasmid 1-3,

DNA. Lanes 3,6

samples

digested

with

139 and 1614 to yield a 1475-bp

Lanes 4-6, samples digested with Hind111 which has sites at nt

1007, 1711, 2768 and 4085 to yield 704, 1057 and 1317-bp respectively.

fragments,

Lanes 7-9, samples digested with XhoI which has sites at nt

2297 and 3184 to give a 887-bp

fragment.

flanking

or plasmid.

sequences

in the genome

Higher

Fig. 4. Construction of the disruption of the rad3 gene. The 1057-bp Hind111 fragment of pSub2.6 which corresponds to nt 1711 to 2768 was inserted

into the Hind111 site of the Sz. pombe integrative

(Sunnerhagen

et al., 1990) to create the plasmid

vector pLEU2

pR3H l.OLEU2.

with the 4.5kb

all of the rud3 insert and

1, 4 and 7 are Sz. pombe 972 h-

Lanes 2,5 and 8 are pSub2.6

and 9 are pSubl.41

enzymes were separated

which contains

vector DNA.

are all

of Sz. pombe genomic and

by digestion

DNAs with restriction

SmaI-Sal1

0.5 kb of flanking

and the Sz. pombe genome

pSubl.41

leu'h

M, bands

represent

Two independent integrants (dR3-1 and dR3-5) were crossed to a h+ rad3-136 leul-32 Ural strain and segregation of the rad, leu and his markers was followed by random spore analysis. All 216 spores analyzed from each cross were Rad- . In both crosses, the Zeu and his markers segregated independently of each other. This demonstrates that pR3H 1.OLEUZ had indeed integrated at the rad3 locus by homologous recombination. Similar results were obtained in another cross in which the integrant strain dR3- 1 was used as a mating partner for a h+ leul-32 (rad+) strain. (d) Sequence of the rad3+ gene The sequence of the rad3 + gene (Fig. 5) has a high A+T content (64%) which is characteristic of Sz. pombe DNA.

Analysis of the sequence shows a single 3210-bp ORF extending from nt 331 to nt 3543. Computer analysis (Staden, 1984) of the above ORF suggests a high likelihood of a protein-coding region. Translation of the 3210-bp ORF yields a 1070-aa protein with a calculated M, of 121974. Sharp et al. (1988) have shown that in Sz. pombe the codon bias of weakly expressed genes is not only weaker than that of highly expressed genes, but that weakly expressed genes show a preference for those codons in degenerate sets that are not preferred by their highly expressed counterparts. Another pattern in degenerate codon sets is that the bias is in favor of G or C nt at the third position in genes with a high CBI, while the bias is in favor of A or T nt at the third position in low CBI genes (Russell, 1989). The putative Rad3 protein follows both patterns for weakly expressed genes and has a calculated CBI of -0.01. One other ORF is detected within the sequenced region: a 694-bp stretch found between nt 3524 and 4217. No splice junction (Mertins and Gallwitz, 1987) is evident between the 3210-bp ORF and the 694-bp ORF. It is unlikely that the 694-bp ORF is required for rad3 complementation because pSubl.41, which complements rad3-136, terminates its 3’ end between nt 4000 and 4100 (data not shown).

~GAAGAGGAATTGGGATAGACGTTCTGCCATTGCAAGGAGAGAACC~TTATATGTC~

KKRNWDRRSAIARKENRYMS XhOI ClaI c,TGGAAGATGCTACCTSTCGAGAATCATCGATCTCRRAA VEDATSRESSISKVES

CTTGATTTTTAATACGTTGAAAATATTGGCGGTGGAAAAC~TCA .XbaI TCTTCCTGGRTCTCCTTATCTAGAGTGGTAGARGAAGRAGTTACATTTTGTACTATTG

L

F

s

R

rTTCCTTC~CATTAGGTATTGTCTCTTTRRATTGTGGATTT~ATG~T=GTG~ATT~ FPSKTLGIVSLNCGFHARAL

GARGTTATATCTTCTCTGATAAACAGCGGAATATTT*ATC*~~CATT~~TCTCA~C~CT .Hl”I CTGCRACAAATT‘CCTCGACGCGTCATATATCCGTTTGGCCTCCATATTG~

CCRI\CAGTGTCC‘TTGCGATTGTCCAAGGTATGGGT AALAACCGAACAT*GCC*GTTTA MGKKPNlASL TTTGCTCAGCTTATGAATATTTCCGAGGGCGATTTTCTTATTCG~CACAGGCGTACAC~ DFLIRTQAYT FAQLMNISEG TTACCATTCCTTGTACTTACTAAARACARAGCGTTRRTAGTCA KALIVRTAELS LPFLVLTKN CAAAGTGATGTTGCTACTTTGTGCCTTACCAATATGCATATCCTTGCTTCGCTACTT QSDVATLCLTNMHKILASLL

~\GGCGRAAATCGGTTTGCTTRI\CAGCATGCTGCAATCG K A K I GLI.NSMLQSG”YESL”

ACTACGGATCATCCTAATTTGGAAGAGTGTGTGATGCTTCTTCTTTCACTGGCCACTTCT TTDHPNLEESVMLLLSLATS

TTGAGTTTAGATTCTTTTATRRTCAATGACRRCCACGAGTATTCGAAGA~GTT~TTT~ LSLDSFIINDNHEYSKNLNL Hind111 GGTATTGAAGCTTCATGGCGTTCGCTATCTATTGATTCGT GIEASWRSLSIDSLKKCLSK

GATTTT‘AARAAGTTGATTTRRCGTCTTTGTTACGCTCTG~TG DFEK”DLTSLLRSDPISIT” GAtTTGTTACAGCTTTATCAGAATGATGTTCCTCRTG ELLQLYQNDVPHEKIENALR

&AAATTGAAA~TGCTTT.&

CTTTATGATTTTTTTAATAATCACATTTTCCGTATCTTRGTATCCTT LAEFSNlL LYDFFNNHILGI XnmI.

AliCGACCTGAAAGGRAAGACTTCAATTAATGAAAAGATTAAGAC~TTGTCGGCATTG~ NDLKGKTSINEKIKTIVGIE

ATTGTTCTTCGCCGTC‘GCTTAGCCAAGTAGCTCCGTACG IVLRRRLSQVAPYGKFKHQI

TTGATATTAGCAACCRAGGAGCCCGRGTATRGTTCRATTG~T‘GTTT~~TCTTGT~T~ LILATKEPEYSS IAGLSLVI

RGTCTGGATTCAGCGTGCTTTAGCCTTAAGGCTAAAGARA SLDSACFSLKRKEIFCS

L

Q

N

ATGTTTGATTTGGTTGATGAGCI\TGAAGAAAGI\CCTA

KFDLVDEHEERPXNRKETLG AATCCACTTAAAGGAAAAGTGTTCTTGAAACTTACAAAATGGCTCGGAAAAGCTGGCC~

NPLKGKVFLKLTKWLGKAGQ

GTTTGTTATTTAG‘TTTACAARAATTAGAACTTTTTTTTCAAG~=AAGG~‘GA~GAGTT~ v c Y LGLQKLELFFQAKVDEL

CTGGGATTGkPGGATTTGGAGACGTATTATCATRAAGCGGTAGAGATTTACCTCAG~T~

LGLKDLETYYHKAVEIYLRM

CATGACACACTAAATTTGGACATATCCAACGAAGTTCTGG‘ATG~~T~ HDTLNLDISNEVLDQLLRCL

TGAGI\ATACGCATTATTATCTTGGCCATCATCGAGTTTT~TGTATG~G~G~C~ f

TTAGATTGTTGTGTRAAATATGCTTCAA=~~TATGCAAA LDCC”KYASTNMQlSYI.AAX

GCTCCCAGTTRATGAACAGAGCGRACGATTTTTAAGT‘GTGAGTTAGT~~T=G=AT~~ TAACGRATTTGGTC‘ATCTTTGTRCTATGGTACAAATCAT XnmI ATTGCTCACACTGTGGCTTGATTTTGGGGCCGAAG4ACTTCGCTTAT~T~GATGACGG CGAAAAGTACTTTCGTGAACACATTRTCTCTCTTCGAG AAALAATCTTTGGAA~TTATGAA

TTTATGCAATCGCAGTTRATTCCAGCTTTCCTTGTTA=TACTGATA=T~AG=A=AAGG~ FMQSQLIPAFLVTTDTKAQG .HindlII TTT~TTGCCTATGCTCTGCAAGAGTTTCTAAAGCTTGGTGGT~~G~AGTGAT~ FLAYALQEFLKLGGFKSAVI

TTCGRATGTTTGTCGCCTTTCTATGRAAATTCCTCAATACTTTTTTCTGGTTGCATTAT~ CC~TGATATCCAGAGTATGCCATCCAAATMTlVULGTTTAT~TTTTGG~CATA~ AATTGCAAI\CGTTGTAGCATCTTATCCTGGGGAGACGCTT -TCGACTTCTCRRAAGCGCTCGCTTCGTGGAAAAAGTT= HindIII TAGG~TCTATGTCTTCCRAAGTTGRTRTAAAAGCTT~T

AAACGTGTGCTTATACCATTTTTAACTTCCA1\GTATCAT~T~CACC~~CCCC-T~ K R v L I PFLTSKYHLTPIPKI

CATTACTGAAAAGTTAATCRATTTGTGCAATACAAGGATT~CAGT~TCTGT~~

GAcATTCGGTACCCTATTTATRAAGAAAATGTTACTATTC DIRYPIYKENVTIHTWMQLF

GAGCTTAAAGGATCCAC

to known regions.

internal

sequences

were used to obtain

In both cases, the sequence

from ss or ds templates

by the dideoxy chain-termination

et al., 1977) using 5’-[35S]thio-dATP (United Fig. 5. Nucleotide X63544),

sequence

and the deduced

of the rad3’ aa sequence.

gene (EMBL Plasmid

pSub2.6

with SmaI and Sal1 and a 4.8-kb fragment containing sert was isolated and inserted into the pBluescriptKS

accession

No.

was digested

the Sz. pombe inand SK- vectors

(Stratagene, La Jolla, CA), and sequencing substrates were generated by nested deletions using exonuclease III and mung-bean nuclease (Henikoff, 1987). Alternatively,

ss oligodeoxyribonucleotide

primers complementary

States Biochemical,

were obtained

sequences

from both strands

by sequencing

Cleveland, pSub2.6,

from some

was determined method (Sanger

(Biggin et al., 1983) and Sequenase OH). Positions and pSubl.41

129 through

4214

was used as a tem-

plate to extend the sequence upstream from position 129 using primers homologous to the rud3 sequence. Single-letter aa symbols are given below the second

nt of each codon.

The putative

‘TATA-like’

boxes upstream

from the gene and the putative polyadenylation signal downstream from the gene are denoted in bold letters. Relevant restriction enzyme sites are shown.

88 In Sz. pornbe, transcription usually begins within 200 bp upstream from the start codon (Russell, 1989). The presented sequence extends 330 bp upstream from the coding region. Because Sz. pombe DNA has a high A+T content, transcriptional start (TATA boxes) and poiy(A) addition signals have not been well defined. No discernible TATA boxes can be identified upstream from the ORF, but two possible TATA sequences exist at positions upstream from potential start codons. One such sequence (TATTTT) can be seen at nt 106 (Fig. 5) and another (TATTTT) can be seen beginning at nt 212. One possible poly(A) signal (AATAAA) is seen at nt 3932, and this is 390 bp downstream from the end of the ORF. In eukaryotic cells, the first available ATG in an ORF is almost always used as the start codon (Kozak, 1989). The first in-frame ATG in the rud3 ORF is located at nt 331 and the next in-frame ATG is seen at nt 373. The sequence surrounding the first but not the second ATG conforms to the consensus sequence for start codons derived by Kozak (1989), and is therefore likely to be the correct start codon. Translation beginning at the first ATG would result in a polypeptide with an &fr 121974 while the polypeptide resulting from translation initiating at the second ATG would be of M, 117 663. A number of proteins involved in DNA repair and recombination have shown a high degree of functional and structural conservation between budding and fission yeasts and also between yeast and higher eukaryotes (Subramani, 1991). Consequently, searches for genes homologous to rud3 were undertaken. Functional complementation of the SZ. pombe rad3-136 mutation with plasmids containing cloned copies of the S. cerevisiae genes RADl (Yang and Friedberg, 1984), RADZ (Naumovski and Friedberg, 1985), RAD3 (Montelone et al., 1988), R4D6 (Jentsch et al., 1987), RADlO (Weiss and Friedberg, 1985) and RAD52 (Adzuma et al., 1984) failed to increase the level of UV resistance above background (data not shown). Several other SZ. pombe genes (radl, rad9 and radl7) involved in G2 arrest and in the coupling of mitosis and DNA synthesis (Rowley et al., 1992; Al-Khodairy et al., 1992) also failed to complement the rad3-136 strain (G. Jimenez and S. S ., unpublished data). Searches of existing protein databases (NBRF version 25.0, SwissProt version 16.0) and the nt sequence databases (EMBL version 26.0) failed to detect any structural similarities. The Rad3 aa sequence was also screened for the presence of a number of domains associated with proteins involved in DNA repair and recombination. These include ATP-binding domains, pu~ne-binding domains, domains specific for helicases, or ATPases, and regions conserved between ubiquitin-conjugating enzymes. In no case was a related sequence found in the Sz. pombe rad3 sequence.

(e) Phenotypic analysis of a strain in which the ru83 gene is disrupted Fig. 6 shows the survival of the integrant dR3-1 in response to UV- and y-irradiation. The dR3-1 strain was somewhat more sensitive than the rad3-136 mutant to UV light (Fig. 6A) and less sensitive to y-radiation (Fig. 6B). (f) Subchromosomal localization of the rud3 gene The rad3 locus has not previously been assigned, genetically or physically, to a position in the Sz. pombe genome. Fan et al. (1989) have established a NorI restriction map of the complete Sz, pombe genome. When the pSub2.6 insert fragment was used to probe NotI-digested genomic DNA, it hybridized to a 1500-kb fragment (data not shown). This corresponds to NotI fragment C found on chromosome II of Sz. pombe.

,107

0

0.00001

’

0

46

’ 20

QO

136

UV Dose

(J/&

I

1

40

60 Dose

180

225

I 80

100

120

140

(krad)

Fig. 6. Survival of the strain with a disruption of the rud3 gene, dR3-1, and the ~23-136 mutant in response to UV- and y-irradiation. Cells were irradiated as described in the legend to Fig. 1. Each point represents the average of colonies in at least two experiments. Squares, 972h.- ; triangles, h- rad3-136 uru4; circles, dR3-1 strain. (A) Survival after UV-irradiation; (B) survival

after y-irradiation.

89 (g) Conclusions (I) We have cloned a gene that complements the rad3136 mutation and shown that this gene is the rad3 + gene. (2) We have physically mapped the rad3 + gene to NotI fragment C on chromosome II of Sz. pombe. (3) The cloned rad3 + gene complements all the known deficiencies of the rad3- 136 mutant (UV-sensitivity, y-ray sensitivity, G2-arrest deficiency and hydroxyurea sensitivity; see Fig. 1 and Jimenez et al., 1992). (4) We have sub-localized the rad3’ gene to a portion of the plasmid pSubl.41. (5) The rad3 + gene consists of an intronless ORF of 32 10 bp (1070 aa) that could encode a protein ofM, 121974. (6) The gene is poorly expressed with a CBI of -0.01. (7) The rad3 + gene is a new gene with no obvious structural motifs or close homologs.

Jentsch,

S., McGrath,

gene RAD6

J.P. and Varshavsky,

encodes

A.: The yeast DNA repair

a ubiquitin-conjugating

(1987) 131-134. Jimenez, G., Yucel, J., Rowley,

R. and Subramani,

of Schizosaccharomycespombe tions and in DNA

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