Cloning and characterization of the Schizosaccharomyces pombe DNA ligase gene CDC17

GHW. 41 (19X6) 321-325

321

Elscvier GENE

1518

Cloning and characterization (Recombinant

DNA;

L.H. Johnstona*,

of the Schizosacchavumyces

fission yeast;

suppressor;

promoter;

pombe DNA iigase gene CDCZ7 with Sacckarnm_vces cereviskre)

complementation

D.G. Barkera and Paul Nurseb

‘ILaboratrw.vof CeN Propagation, National Institute for Medicat Resecrrch, The Ridgewq. Mill Hill, London NW7 IAA, Tel. (01) 9593666, and ’ CeN Cj’cle Control Laborator?,. Imperial Cancer Research Fund, Lincoln k Iun Fields. Loudon WCZA 3PX (Ci~k’.J Tei. {01)2420200 (Received

July jth,

(Revision

received

(Accepted

1985) October

November

21st, 1985)

25th, 1985)

SUMMARY

The Sc~iz(~sacc~aromyces pombe CDCI 7 gene has been cloned by complementation of the cdel7 mutant coding for temperature-sensitive DNA ligase. An allele-specific suppressor active only in the presence of a high osmotic pressure was also isolated. The cloned CDCI 7 gene failed to complement the analogous DNA ligase mutation, cd&, in Saccharomyces cerevisiae, although the reverse complementation was successful [Barker and Johnston, Eur. J. Biochem. 134 (1983) 315-3191. The CDCl7 gene specifies a 2%kb transcript. __

Johnston,

INTRODUCTION

1983), and the analogous

gene CDCl7

from the fission yeast S. pombe (Nasmyth, 1977). The availability of ts mutants in these genes and of

The ubiquitous enzyme DNA ligase is required principally during DNA synthesis for the joining of Okazaki fragments and also for the repair of damaged DNA. A study of the control of expression of DNA ligase in different organisms might reveal features of cellular regulation which have been conserved during evolution. With this in mind we have initiated parallel studies into the regulation of the DNA ligase gene CDC9 from budding yeast, S. cerevisiae (Johnston and Nasmyth, 1978; Barker and

plasmid vector transformation systems for both organisms together provide a convenient approach both to cloning the genes and also to examining their heterologous expression and regulation. Following the isolation of the S. cerevisiae CDC9 gene (Barker and Johnston, 1983) we now describe the cloning and preliminary characterisation of the S. pombe CDCl7 gent.

* To whom correspondence

EXPERIMENTAL

and

reprint

requests

should

be

AND

DISCUSSION

addressed. Abbreviations:

bp, base pair(s);

types; kb, kilobase reading

plasmid-carrier

037X-I 119:X6/503.50

, S. pombe mating

or 1000 bp; nt. nucieotide(s);

frame; ss. single-stranded;

designates

h + and h

0

ORF, open

ts, temperature-sensitive:

state.

19X6 Elsevier

Science

Publishers

[ ],

(a) Cloning of CLlCZ7 A cdcf 7-K42 leui-32 strain of S. pombe was transformed (Beach et al., 1982a) with DNA from two

B.V. (Biomedical

Division)

322

clone banks, one in vector YEpl3 (Russell and Hall, 1983) and one constructed by inserting a partial Hind111 digest of chromosomal pDB262

(a positive selection

DNA

37°C after replication from the original sorbitol-agar plates. S5 tr~sformants also behaved similarly when first replicated onto minimal agar plates and

into vector

vector based upon the

bacteriophage

1. ci gene). Plates were incubated

the permissive

temperature

transformants

were replicated

grown at 25°C before transfer to YE-phloxin at 37°C. Only when replicated onto another sorbitol-

at

agar plate, which contains

of 25 “C and the resulting to yeast-extract

transformants

agar

plates containing 20 [kg phloxin B/ml YE-phloxin for further incubation at the restrictive temperature of 37°C. The phloxin stains dead cells a deep red colour

and therefore

aids the selection

did all

grow at 37 ‘C. The SS complementing

activity therefore differed from that of SlO and H2 in appearing to require high osmotic pressure for full effect; moreover,

of transfor-

it alone was alleIe-speci~c,

allele c&I 7-M75 being unaffected

mants that are able to grow at 37’ C and have a paler colour. A total of 1.5 colonies were obtained

1.2 M sorbitol,

another

by S5. Clone 55

therefore appears to be a suppressor and may be a protein which normally interacts with DNA ligase

at 37 “C

by this means, all of which showed the instability characteristic of plasmid-borne genes in yeast. Recombinant plasmid DNA extracted from three of these colonies and designated S5 and S 10 (vector Y Ep 13) and H2 (vector pDB262) was used to transform E. coli (as in Barker and Johnston, 1983). When re-isolated, each plasmid transformed S. pombe cdcl7 to growth on sorbitol-agar plates (Beach et al., 1982a) at the restrictive temperature with high frequency (about 4-6 x lO’/iig DNA). However, unlike SIO and H2 transformants, S5 transformants required some 44 h preincubation at 25’ C before the maximum number of colonies was observed at 37 ’ C, and only 2-3 T<,of these transformants were able to grow on YE-phloxin plates at

and which can partially overcome the cdc17-K42 mutation when its gene is present on a multi-copy plasmid. (b) Mapping of the CLXl7

gene

Restriction analysis (Fig. 1) and sub-cloning showed that S 10 and H2 had some 4.5 kb in common and that the CDC17 gene, possibly together with the information necessary for its expression, lies between the leftmost EcnRI site and the left BgtII site. Southern hybridisation (Southern, 1975) studies with the two probes shown in Fig. 1 confirmed that neither of these two clones had any sequence in common with S5 and that they were unrearranged

+.---+

lkb

2.4 kb Probe I

I

SSEAP I I

E

EACE

Bg

E

II

6.7 kb Probe I

I

HE I

H2

SSEAP I I III

E I

EAC E I II I

Bg

E

I

I

Bg I

C

H

I

t I

1

L

I

5’ SI Probe Fig. 1. Restriction used in Southern indicate

map ofclones hybridisation

complementing experiments

the ends of the c‘DCI7 transcript

U~irri; Bg, B@II; B, Ba/rtHi.

3’SI

cdc17.The hatched

areas represent

are shown. The two S 1mapping as determined

bq Sl nuclease

probes described mapping.

Probe

vector sequences

and the 2.4-kb and h.7-kb probes

rn Fig. 3 are also indicated.

H. HitzdIII;

E, EcoRI;

The two :~rows

S. Suil; A, Awl;

P. Purl: C.

323

compared (which

with

their

chromosomal

in S. pombe

are present

(c) Ligase synthesis

homologues

DNA

as single

copies) (not shown).

nation strain

into a cdcl7-K42

transformed

leul-32

the unrelated

h

comparative

and a stable clone from each was isolated.

These putative

integrants,

which could both grow at

plasmids,

not do so. For

the entire Hind111 fragment into YEpl3

(here-

so that all the cloned DNAs

in the same vector.

S5, SlO and HZ-YEp13,

The three

were each trans-

formed into cdcl7 and the crude extracts prepared from these transformants were assayed for levels of DNA ligase. S5 transformants possessed only the same low levels of a ts activity found with cdcl7 alone (Table I), confirming that S5 is probably a suppressor of cdcl7. In contrast, H2-Y Ep 13 directed the synthesis of a temperature-stable ligase that was present at levels some lo-fold above that of the wild type, presumably reflecting the plasmid copy number. The remaining plasmid, SlO, also specified a temperature-stable enzyme, but in this case the activity was present at only 80% of the wild-type level, possibly reflecting the lower stability of this plasmid as compared with H2-YEp13 (94”/, loss

in both crosses while no ts clones

served out of 500 spores analysed for each integrant, so LEU + was very closely linked to CDC17 and the S 10 and H2 plasmids had integrated at the site of the CDC17 gene.

Assays

in S5 should

after called H2-YEp13), could be compared

were detected in 300 spores from each cross. Thus the marker allowing growth at 36’ C in the integrants was closely linked to CDCI 7. To prove that the plasmid sequences were associated with the CDC17 site the original integrants were crossed to a cdcZ7-K42 leul-32 h’ strain. No recombinants were ob-

TABLE

insert purposes

from H2 (Fig. 1) was subcloned

36°C were crossed to an ade6-704 h’ strain. Random spore analysis showed that the ade6 marker freely recombined

to code for a DNA

ligase (Nasmyth, 1977), plasmids H2 and SlO should both direct the synthesis of this enzyme, while

should occur at the CDC17 locus. They were

therefore

as CDC17 is known

Finally,

If plasmids H2 and S 10 do carry the CDC17 gene, integration into the genome by homologous recombi-

I of DNA ligase in crude extracts

(A) Cells grown

at 25’C

and switched

from cdcl7 to 37’C

transformants

2 h before harvestmg

and assay

at 25’C

Ligase activity” cdcl7

Wild type

cdcl7

cdcl7

25’C

0.10

0.03

0.04

0.085

0.95

37’C

0.30

0

0

0.12

2.17

(B) Crude

extracts

prepared

from cells grown

cdcl7

[S5]

[SlO]

at 25’C

were incubated

[St01

cdcl7

[H2-YEpI

at either 25’C

or 34°C for 5 min before assaying

at 25-C

Ligase activity il Wild type

I

25’C

0.1 -

34°C

0.07

3 The assay measures with ‘*P, annealed

0.08

0.64

0

0.05

0.44

the ligation of two short oligodeoxynucleotides

[HZ-YEp13]

(15-mer and 17.mer Ml3 sequencing

on M 13 ss DNA (Barker et al., 1985). The product

The position

band and determining

1.5~mer, 0.6 ng labelled

cdcl7

0.03

contiguously

and gel electrophoresis. appropriate

cdcl7

of the product

the amount

is located

by autoradiography

of label. In this experiment

17-mer and 1.O )_tgcrude cell extract

is separated

was assayed

primers),

from unreacted

and the assay

each assay contained

one 5’ end-labelled

substrate

is quantified

by denaturation

by cutting

out the

400 ng M 13 ss DNA, 2 ng unlabelled

for 4 min and results are expressed

as fmol of substrate

ligated.

324

compared

with 60”/

non-selective

loss after ten generations

a

of

growth),

In summary,

b

c

we have cloned a gene which comple-

ments cdcl7, integrates back into the genome at the CDCI 7 locus and which directs the synthesis of a DNA ligase. It is therefore and S 10 do contain (d) Identification

clear that plasmids

H2

the CDC17 gene.

and mapping of the CDCl7

Total RNA extracted

from exponentially

to Northern

hybridisation

1.23

tran-

O-84

growing

O-57

script

cells was subjected

1.65

anal-

ysis (Fig. 2). The ~~~zdIII-~g~II fragment, common to both SlO and H2, hybridised to two RNA species of 1.6 kb and 2.8 kb (Fig. 2, lane a), Subcloning experiments (see above) had shown that CDCI 7 spans the DNA between the leftmost EcoRI and &$I1 sites (Fig. 1) and since the PstI-CIaI fragment from within this region only hybridised to the 2.8-kb transcript (Fig. 2, lane b) then this must correspond to the CDCl7 mRNA. A probe prepared from part of the 880 bp of DNA to the left of the Hind111 site in SlO (Fig. 1) only hybridised to the 1.6-kb transcript (Fig. 1, lane c) which must therefore be derived from an adjacent gene. S 1 nuclease mapping of the CDCf 7 transcript was carried out by cloning into the polylinker site of

Oe36 Fig. 3. S I

nucloasemappingofthe

5’ end and 3’ polyadenylation

site of the CDCI7 gene. Appropriate ning the 5’ and 3’ ends ofCDCI7 cloned

into the bacteriophage

restriction

fragments

vector

hll3mpl

1 (Norrander

ct al.. 1983). Total S. porn/x RNA WDSthen hybridised with an excess of ss preparations M”,,

forrnanidc.

nt&xc:ml

for

The hybrid

was digested

1 h at 30-C under standard

a neutral

blotting.

l.anc c shwvs

agarox the

at 42‘C

of this RNA in the presence with 200 units conditions

et al., 19X2), and the protected DNA fragments through

span-

(see section d and Fig. 1) wcrc

gel and single

identified 5’-protected

of SI

(Mamatis

electrophoresed by

Southern

fragment

of

550 _t 50 nt and lane b shows the sin& 3’-protecrcd fragment of 600 + 50 nt. DNA fragments of known M, arc shown in lane a, the values given being in kb.

abed M 13mpl1, the 1.6-kb EcoRI fragment and the 2.6kb CIUI fragment, which should span the termini of thegene (see section b above, and Fig. 1). Total yeast

l-65

c

le23 Fig. 2. Identification extracted

of the CDCl7

from S. porn/w. subjected

transcript.

and then trnnsferrcd

to a nitroccllulose

198).

this membrane

After cutting

hybridised

with different

Total

‘*P-labeiled

membrane into strips,

(Aves et al., each lane was

DNA probes from the S 10

clone (Fig. 1) as follows: (a) the 4.2-kb NindIII-Bg/II (b) the l.Mb

Psrl-Cl01

(c) an internal

fragment

Hi~dlll

fragment

RNA was

to agarose gel electrophoresis

fragment;

from within the CDC17 gene;

from the 880-bp DNA to the left of the

site: (d) DNA f’ragmenrs of known /M,, the values given

hcing in kb,

RNA was hybridised to ss DNA fragments of appropriate orientations, and after nuclease digestion the protected fragment of the EcoRI subclone was found to be 550 + 50 bp and that of the ClaI subclone was 600 2 50 bp (Fig. 3, .lane c and b). The orientation of the inserts in these two phage DNAs, taken together with preliminary DNA sequence analysis of part of the CDCI 7 coding region indicated that transcription initiates within the EcoRI fragment and terminates within the C/u1 fragment (Fig. 1). This would give a total transcript size [without poly(A)] of 2700 nt, which is in good agreement with the value of2800 nt obtained by Northern hybridisation analysis and consistent with a M, of 90000, which has been determined for the S. ~(~~7be ligasc subunit (G. Banks, manuscript in preparation).

325

(e) CDCl7

does not complement

We have previously

S. cerevisiae

shown that the cloned gene for

DNA ligase CDC9, from the budding siae, is expressed cdcl7 mutations

cdc9

in S. pombe

and

yeast S. cerevicomplements

(Barker and Johnston,

1983). How-

ever, neither the H2 nor S 10 clones of CDCZ 7 were able to complement

cdc9. The most

ation for this is lack of transcription transcripts Northern

could not be detected analysis.

likely explansince CDCI 7

in S. cerevisiae by

A similar situation

exists between

the S. pombe CDC2 and S. cerevisiae CDC28 genes (Beach et al., 1982b) and these failures

of comple-

mentations may simply reflect differences between the promoters of the two organisms. The identification of the CDC17 gene and its transcript should now form the basis for further studies on the regulation of CDCI 7 both in the mitotic cell cycle and in response to DNA damage.

REFERENCES Aves,

S.J., Durkacz,

quencing

A. and Nurse, control

cdcl0 “start”

mycespombe

Barker,

B., Carr,

and transcriptional

D.G. and Johnston,

a structural

P.: Cloning,

gene. EMBO J. 4

( 1985)457-463.

L.H.: Succharomtws

wreviritre

gene for DNA ligase which complcmenta

succharomJ~ce.v pomhe

Eur. J. Biochcm.

cdc17.

be-

of the Schi~[-ovtrc,~hclro49. Schko-

134 (1983)

315-319. Barker,

D.G., Johnson,

A.L. and Johnston,

assay for DNA ligase revcala in cdc9 mutants Gcnct.

L.H.: An improved

temperature-sensitive

of Scrcchawnws

activit) Mol. Gen.

cerrvivicw.

200 (1985) 458-462.

Beach, D., Durkacz,

B. and Nurse.

P.: ConstructIon

pombe gene bank

of a S&i:+

in a yeast bacterial

shuttle

vector and its use to isolate genes by complemcntation.

Mol.

sacchuromwes

Gen. Genet.

187 (1982a)

Beach, D. Durkacz, cell cycle control 300 (1982b) Johnston,

326-329.

B. and Nurse. P.: Functionally genes in budding

homologous

and fission yeast. Nature

706-709.

L.H. and Nasmyth,

cycle mutant

cwevititrr

cdl

ligaae. Nature

274

K.A.: Succharomycrr

cdc9 is defective

in DNA

(1978) 891-893. Maniatis,

T., Fritsch,

A Laboratory Spring Harbor, Nasmyth,

ACKNOWLEDGEMENTS

K.A.:

structural

We would like to thank Don Williamson cal reading of the manuscript.

for criti-

E.F. and Sambrook,

Manual.

Cloning.

Laboratory.

Cold

NY, 1982. Temperature-sensitive

lethal

mutants

in the

gene for DNA ligase in the yeast S~hi-r,srr~~lttrro-

myres pombe. Cell 12 (1977)

Norrander,

J.: Molecular

Cold Spring Harbor

J., Kempe.

11OY-I 120.

‘T. and Messing,

J.: Construction

of im-

proved M 13 vectors using ohgodeoxyribontxlcotide-directed mutagenesis. Russell,

Gene 26 (1083)

dehydrogenase ,,l~w.v /x&w. Southern,

101-106.

P. and Hall. B.D.: The primary gent

separated

of specific

sequcnccs

by gel electrophoresis.

(1Y75) 503-517. Communicated

of the alcohol Scl,i-o.st/cchrrrt,-

J. Biol. Chcm. 258 (1983) 143-149.

E.M.: Detection

fragments

structure

from the fission yeast

by K.F. Chater.

among

DNA

J. Mol. Biol. YX