© 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 18
4913-4918
The 66 kDa component of yeast SFI, stimulatory factor I,
is hsp60
Jean K.Smiley, William Clay Brown and Judith L.Campbell*
Braun Laboratories, Division of Chemistry, California Institute of Technology, Pasadena, CA 91125,
USA
Received April 29, 1992; Revised and Accepted August 12, 1992
ABSTRACT
DNA polymerase e stimulatory factor I (SFI) has been
shown to contain three peptldes, p66, p37 and p13.
Two of these components have been Identified. The
p66 gene was cloned by using a p66 antibody to screen
a Xgt11 library. A portion of the gene was sequenced
and confirmed to encode p66 by the presence of
protein sequence corresponding to that of p66 tryptic
peptides. The gene was identified as HSP60 by a
homology search of GenBank. Tryptic peptides of p37
were sequenced and identified as belonging to yeast
translation initiation factor 4A by a homology search
of PIR. The HSP60 gene maps to chromosome XII.
INTRODUCTION
DNA replication is a complex process that is essential for cell
viability. The timing, fidelity, and extent of DNA replication must
be accurately controlled. The central biochemical reaction to
DNA replication, the copying of the parent template DNA into
a new polynucleotide chain, is performed by DNA polymerases.
Yeast encodes three DNA polymerases, a, 5, and e, that are the
products of genes necessary for cell viability (reviewed in 1,2).
This implies that they are involved in DNA replication.
The activity of the DNA polymerases is controlled by accessory
proteins. RF-A, a single-stranded DNA binding protein, RF-C,
a single-stranded DNA dependent ATPase, and PCNA, a
processivity factor, together stimulate and control the activity of
DNA polymerase 8 (3-6). They also stimulate DNA polymerase
6 (5,7). The involvement of these three stimulatory factors in
DNA replication was first demonstrated in the SV40 in vitro DNA
replication system (reviewed, in 8-10).
In the absence of an efficient yeast in vitro DNA replication
system, new yeast DNA stimulatory factors must be identified
by alternate methods. During the purification of DNA polymerase
t, two stimulatory factors were identified (11). One of these
factors, stimulatory factor I (SFI), has been purified (12). It is
an apparent complex containing three proteins of molecular
weight 66, 37, and 13.5 kd. The large protein has single-stranded
DNA binding activity. This complex does not enhance
processivity. In order to test whether SFI or any of the
* To whom correspondence should be addressed
components of SFI are bona fide replication proteins, we
undertook to isolate the genes encoding p66 and p37.
Determination of amino acid sequence revealed that p37 was
translation initiation factor 4A (T1F1), the yeast homolog of
initiation factor eIF-4A. Cloning and sequencing of p66 revealed
that it is encoded by HSP60 (heat shock protein 60).
MATERIALS AND METHODS
Strains and media
Bacterial strain XLl-Blue (Stratagene) was used in cloning. p66
was purified from protease-deficient strain PEP4D [Mate/a
hisll+ trpl/+ prcl-126 pep4-3 prbl-1122lprbl-1122 canll
canl] (13). The Xgtll library was grown in Y1090 (14). E.coli
strains RDP146 and NS2114Sm were used in the Tn3 insertion
mutagenesis (15). The p66 gene was disrupted in S.cerevisiae
strain SEY6210.5 [Matala leu2-3, 112/leu2-l,112 ura3-52/
ura3-52 his3-200/his3-200 tgrpl-901lirpl-901 Iys2-8O1I +
ade2-101l+ suc2-9/+ mel-lmel- (S. Emr. umpublished)].
Bacteria were grown in LB (16). Yeast were grown in YPD
or plated on YPD or SC-his plates (16). Diploid strains were
sporulated on sporulation plates (17).
Antibody production
p66 (fraction V and the flow-through from the hydroxylapatite
column (12) was purified for injection by SDS-PAGE (18). The
band containing p<56 was cut out and prepared for injection (19).
Antiserum was raised in a young female New Zealand White
rabbit, 50 /tg of gel purified p66 in Freund's complete adjuvent
was injected at multiple intradermal sites. The first boost was
after 1 month with 50 \i% p66 in Freund's incomplete adjuvent.
A second boost was two weeks later. Serum was collected 10
days after each boost.
Immunoblots
Proteins were separated by SDS polyacrylamide gel
electrophoresis (18). They were transferred to nitrocellulose using
the method of Burnette at 60 volts overnight in the cold room
using Bio-Rad Trans-Blot cell and Model 250/2.5 power supply
(20). The filter was blocked, probed, and stained (21).
4914 Nucleic Acids Research, Vol. 20, No. 18
Screening of Xgtll library
1 xlO 7 recombinant clones of S.cerevisiae Xgtll library from
Clontech was screened by a polyclonal antibody against p66 (21).
The antibody was used at a 1:100 dilution. Positive clones were
rescreened and plaque purified. To verify the identity of these
clones, oligonucleotides encoding tryptic peptides of p66 were
hybridized to plaque-purified X DNA. To do this, restriction
digests of the X DNA were separated on an agarose gel,
transferred to Genescreen (NEN) by the manufacturer's
recommended conditions, and probed with kinased
oligonucleotides in the presence of tetramethylammonium
chloride (TMA) (22). The blots were prehybridized for 1 hour
and hybridized for 48 hours at 50°C. After hybridization, the
blots were washed for 3 X 30 minutes in 3M TMA containing
50 mM Tris-HCl, pH 8.0 and 0.2% SDS at room temperature
followed by a 1 hour wash in the same buffer at 50°C. The blots
were rinsed in 0.2XSSC and exposed damp with a screen
(Cronex) to XAR5 film (Kodak).
The oligonucleotides were as follows:
JKS1 5'-TATGGallGATCATTTTGC-3'
C
G C C
(sense to a portion of tryptic peptide 1)
JKS2 5'-GCallCCallGAACCallCCallGC-3'
G
(sense to tryptic peptide 2)
Tn3 insertion mutagenesis
H1S3 insertions into the p66 gene were generated using the Tn3
insertion mutagenesis system (15,23). This procedure inserted the
HIS3 gene randomly into the X insert containing the p66 gene
cloned into plasmid pHSS6. Insertions into the cloned X insert were
identified by restriciton analysis using the restriction endonuclease
Notl. Because it was difficult to isolate plasmid DNA from strain
NS2114Sm that contained the final transconjugates, minipreps of
the NS2114Sm transconjugates were used to transform XLl-Blue
cells. Restriction analysis of the transconjugates was carried out
using standard procedures (16).
Integration into the yeast genome
Notl digests of the transconjugates were transformed into strain
SEY6210.5 using the lithium acetate transformation procedure
(17). The presence of an integrated copy was verified by Southern
analysis of yeast genomic miniprep DNA using the Xbal-EcoRI
fragment as a probe. The site of insertion of the HIS3 gene was
determined by restriction mapping using standard procedures
(16).
Diploid strains containing the disrupted p66 gene were
sporulated and dissected by standard procedures (17).
PCR conditions
Primers were prepared on a Pharmacia Gene Assembler. Primers
used were as follows:
JKS5 5'-GCaUGGaUGGTTCallGGallGC-3'
C
(antisense to oligo 2)
JKS6 5'-CCallGGallTTTGGallGATAA-3'
C
C
(sense to a portion of tryptic peptide 3)
The target DNA (Xgtl 1 containing insert 26) was amplified
in 10 mM Tris-HCl, pH 8.3 containing 50 mM KC1, 1.5 mM
MgCl2, 0.2 mM dATP, dGTP, dCTP, TTP, 100 pmol each
primer, 1 ng target DNA, and 0.5 fi\ Taq polymerase from
Perkin-Ehner. The cycle consisted of 1 min at 94°C, 1 min at
37°C, 2 min. at 72°C, repeated 35 times in a Perkin Elmer Cetus
DNA Thermal Cycler.
Tryptic peptides sequencing
Protein to be sequenced was transferred to an Immobilon-P
Membrane (Millipore) (24), cut out, trypsin digested on the
membrane, and the tryptic peptides eluted (25). The eluted
peptides were separated by HPLC and sequenced by the
Laboratory of Macromolecular Structure Analysis, Division of
Biology, California Institute of Technology, Pasadena, CA
91125. Three peptides of p66 gave usable sequence.
Peptide
1:
ID
TorE Y G D D F A K
Peptide 2:
A T T E V A I V D A P E P P A
Peptide
A P G F G D N R
3:
Homology searches
Homology searches were performed at the National Center for
Biotechnology Information using the BLAST network service.
The FAST A program was also used. FAST A uses an algorithm
from Pearson and Lipman (26).
Sequencing by Sequenase
Insert 26 was inserted in both orientations into a modified pT7
vector developed in this lab (manuscript in preparation). A
fragment between the Sail site internal to the cloned insert and
a Sail site in the polylinker of the vector was deleted from each
construct to place a portion of coding sequence next to the
polylinker. A primer was synthesized to the region just upstream
of the polylinker (5'-AGCCAAGCTCGATAA-3') and used to
begin sequencing by chromosome walking. Sequencing reactions
were performed using the Sequenase kit from USB.
Pulsed field gel electrophoresis of yeast chromosomal DNA
A blot of yeast chromosomal DNA separated by pulsed field gel
electrophoresis was obtained from Dr. Elizabeth Bertani,
California Institute of Technology.
RESULTS
Peptide sequencing of p37
Tryptic peptides of p37 from the Hap pool (12) were prepared
and sequenced (see Materials and Methods). The three best
sequences are reported in Figure 1. A homology search of PIR
30.0, September 30, 1991 using either the BLAST or FASTA
programs revealed that peptides 1, 2, and 3 had 93, 85, and 100%
identity with the yeast T1F1/2 protein, respectively. Therefore
we conclude that p37 may be a product of either T1F1 or T1F2
(27), a yeast homolog of translation initiation factor 4A. Since
yeast translation initiation factor 4A has a molecular weight of
around 45 kDa (27,28), p37 is probably a breakdown product.
A 45 kDa protein is often seen in preparation of SFI.
Nucleic Acids Research, Vol. 20, No. 18 4915
Peptide
1:
F Y S T Q I E A
TIF1/2:
378 F Y S T Q I E E L
Peptide 2:
TIF1/2:
Peptide
LorG PorH S D I o r C A
P
S D I
A
E N Y I H R NorA
342 E N Y I H R I
3:
TIF1/2:
V I N Y D L P A N
332
V I N Y D L P A N
Figure 1. Homology of p37 with translation initiation factor 4A. Tryptic peptides
were prepared and sequenced as described in Materials and Methods. The sequence
of tryptic peptides 1, 2, and 3 is compared to the protein encoded by T1F1 and
2 (27) by FASTA or BLAST as described in Materials and Methods. The accession
number of T1F1 is X12813.
1
2
3
5 6
J
-200kDa
-92.5
-69
-46
-30
-21.5
-14.3
Figure 2. Immunoblot using a polyclonal antibody to p66. Protein samples, as
indicated below, were separated by SDS-PAGE, transfered to nitrocellulose, and
probed whh antibody as indicated below and developed as described in Materials
and Methods. Lanes 1 and 4: 150 /ig crude extract,; lanes 2 and 5, 1.8 /ig SFI
(glycerol gradient fraction); lanes 3 and 6: 0.24 y% p66 (glyccrol gradient fraction).
Lanes 1 - 3 were probed with polyclonal ot66. Lanes 4 - 6 were probed with
preimmune serum. Markers: Rainbow markers (Amersham) Myosin, 200K;
phosphorylase b, 92.5K; bovine serum albumin, 69K; ovalbumin, 46K; carbonic
anhydrase, 30kDa; trypsin inhibitor, 21.5K=kDa, and lysozyme, 14.3kDa. (*)
Marker lane.
Cloning of p66
A polyclonal antibody to the large component of SFI, p66, was
generated by immunizing a rabbit with gel purified p66. This
antiserum reacted with a 66 kDa protein on an immunoblot of
glycerol gradient purified p66 and SFI and crude extract (Fig. 2).
A slightly smaller band was also seen in the crude extract. This
is probably a degradation product or a different form of p66 as
its intensity varies with the lysate preparation. TTF1 and TTF2
have identical coding regions. The resideues that are not identical
between p37 and TTF1/2 are probably sequencing errors. Since
this serum is specific for p66, it was appropriate to use this
antibody to screen a Saccharomyces cerevisiae genomic Xgtl 1
library obtained from Clontech for the p66 gene. Three positive
clones were obtained. These contained overlapping restriction
maps (Fig. 3A).
Tryptic peptides of p66 were sequenced (see Materials and
Methods) and degenerate oligonucleotides were made
corresponding to two of the sequences. Each of these
oligonucleotides hybridized to the three positive clones (Fig. 2B),
indicating that these genes encoded p66. By repeating this
hybridization with restricted inserts, the coding sequence of p66
on the insert was identified (data not shown).
SFI is essential
Our initial strategy to disrupt the p66 gene to determine if it
isessential for cell viability was to insert a selectable marker within
the coding region. To do this, the cloned inserts were subcloned
into pUC18. Unfortunately, these constructs were not stably
maintained in Escherichia coli, therefore they were not useful
for further work. The Sacl-Xbal, Xbal-EcoRI, and SacI-EcoRI
fragments also could not be maintained in E.coli when subcloned
into pUC18. Therefore, the gene was disrupted by transposon
mutagenesis (15, 23). This method inserted the HIS3 gene by
transposition into pHSS6 containing the p66 insert. Because this
method only requires one cloning step, a minimal amount of
plasmid DNA is used and the p66 containing plasmid was stable
enough to allow us to proceed. Plasmids containing HIS3
insertions into the p66 gene proved stable in E.coli. Restriction
maps were made of twenty-four transconjugates with insertions
into the cloned gene. Two transconjugates with insertions into
an EcoRV fragment contained within the coding region of p66
were integrated into the yeast genome. The presence of this
fragment in the coding region was deduced from mapping the
sites of the oligonucleotides prepared from amino acid sequence
in the gene by PCR, determining the size of the corresponding
mRNA, and determining probable 5' and 3' ends of the gene
(data not shown). The Eco RV fragment was within the gene
regardless of its probable 5' end. This deduction was later
confirmed by sequencing. Diploids containing the p66 gene
disrupted within the EcoRV-EcoRV fragment were sporulated,
and tetrads were dissected. All spores segregated in a ratio of
2 viable:2 inviable. All viable spores were His". Therefore, the
p66 gene is essential.
SFI is HSP60
The cloned insert was stabilized in E.coli by cloning it into a
modified pT7 vector (see Materials and Methods) which had
strong transcription stop signals placed at both ends of the
polylinker to prevent any transcription of the p66 gene in E.coli.
A portion of the p66 gene was sequenced beginning at the internal
Sail site using the chain termination method (see Materials and
Methods). Sequence corresponding to several of the tryptic
peptides was found, confirming that the correct gene was being
sequenced (see Material and Methods). A total of 926 bases were
sequenced. A homology search of GenBank, release 67,
September 15, 1991, revealed that the p66 gene is 99%
homologous to yeast HSP60. The p66 sequence contained a C
at hsp60 base 1134 instead of an A, a C inserted after hsp60
4916 Nucleic Acids Research, Vol. 20, No. 18
Kpnl
FcoRI
EcoRI
Kpnl
26
EcoRI
?
Kpnl
EcoRI
27A
L
EcoRI
Sad
I
EcoRI
Xbal
Xbal/EcoRV
Kpnl
VII,XV-
A
EcoR
Map
Figure 4. Yeast chromosomal blot. Chromosomal DNA from strain YNN295
(Bio-rad) was separated by pulsed gel electrophoresis. Lane 1: Ethidium bromide
stained gel. Lane 2: Chromosome blot probed with a PCR product between oligos
5 and 6. Lane 3: Chromosome blot probed with a PCR product between oligos
5 and 6 and a plasmid containing the POL3 gene.
Scale
I kb
B
bases 1736 and 2019, and a G deleted at bases 2044 and 2053.
Since only one strand was sequenced, these may be sequencing
errors. Therefore, p66 is yeast hsp60.
1 23
_23kb
-9.4
-6.6
m
#•^-4.4
-2.3
-2.0
Hsp60 is on Chromosome XII
Although HSP60 has been studied extensively, the map position
of HSP60 has never been determined. A Southern blot of
S.cerevisiae yeast chromosomes from strain YNN295 (Bio-Rad)
separated by pulsed gel electrophoresis was probed with a PCR
product produced between oligos 5 and 6 derived from tryptic
peptides 2 and 3. The probe hybridized with chromosome XII
(Fig. 5). Since chromosome XII and IV are poorly separated in
this gel, the blot was reprobed with an equal amount of p66 PCR
product and a probe for a gene on chromosome IV, POL3. Two
bands are seen. The top one is present in both blots, indicating
that HSP60 is on chromosome XII.
DISCUSSION
Figure 3. A. Restriction maps of doned DNA inserts encoding p66. Thick checked
bars represent inserts cloned from the yeast genomic Xgtl 1 library. The thin line
represents the genomic DNA containing the hsp60 gene. The black bar represents
the coding sequence of hsp60. The black square indicates the site of the HIS3
insertion in the disrupted gene of TC-4b. Restriction sites in the cloned inserts
are based on sizes of restricted DNA in agarose gels. Restriction sites in the
genomic hsp6O gene are based on the sequenced gene and reported restriction
sites in surrounding DNA (29,30). B. Southern analysis of positive clones. DNA
was isolated from lysates of three positive Xgtl 1 clones containing inserts encoding
the p66 gene. It was restricted with EcoRI, separated by agarose gel electrophoresis,
and transfered to nitrocellulose. The blot was probed with oligo 1 (shown above)
and 2 (data not shown) as described in Materials and Methods.
The 37 kDa component of SFI, has been identified as containing
some sequence identical to yeast translation initiation factor 4A
by tryptic peptide sequencing and a homology search of PIR,
release 30 (28). The p66 gene has been isolated by screening
a Xgtl 1 library with a polyclonal antibody to this protein. The
cloned gene was confirmed to encode p66 by demonstrating that
protein sequence corresponding to tryptic peptides of p66 were
found in the sequenced portion of the gene. By a homology search
of GeneBank, release 67, it was determined that p66 is hsp6O.
The yeast HSP60 gene has been cloned, sequenced, and
demonstrated to be essential for yeast viability (29,30). Strains
Nucleic Acids Research, Vol. 20, No. 18 4917
containing temperature-sensitive alleles have been isolated and
used to demonstrate that hsp60 is a mitochondrial protein
necessary for assembly of macromolecular structures containing
proteins imported into the mitochondria (31). Yeast hsp60 is
homologous to E.coli groEL (29). GroEL is essential for cell
viability and is required for bacteriophage head assembly (32,33).
A 14-mer of groEL monomers folds/refolds proteins by several
similar mechanisms (reviewed in 34-36). Mg-ATP and ATP
hydrolysis are necessary for this process (37). Yeast hsp60 can
substitute, with a loss of efficiency, for E.coli groEL (37).
We do not feel that hsp60 stimulates DNA polymerase e in
vitro by a mechanism involving the oligomeric form of hsp60.
First, the sedimentation co-efficient for the SFI complex is 4.7S
(11). This is much smaller than the S value of 20-25 reported
for the groEL 14-mers that are required for the chaperonin
activity (38,39). This suggests that we have isolated a monomeric
form of hsp60. Second, ATP-Mg is not necessary for its SSB
activity, and ATP is not necessary for its DNA polymerase e
stimulatory activity (11 and unpublished results). dATP, which
is present in the DNA polymerase e stimulatory assays, does not
substitute for ATP in the groEL in vitro assays (37). Finally,
we have not observed an ATPase activity for SFI (11). This may
be due to an absence of K + ions in our assay (40) or to the lack
of the oligomeric structure. Therefore, the mechanism by which
hsp60 stimulates DNA polymerase e in vitro is unknown. p66
has a very weak stimulatory activity on its own (W. C. Brown,
J. K. Smiley and J. L. Campbell, unpublished data). Perhaps
unoligomerized p66 can protect active DNA polymerase from
forming a nonactive aggregate of denatured protein.
These experiments have identified two components of SFI.
However, it is unclear whether these components are involved
in DNA replication or repair. Yeast translation initiation factor
4A is a double-stranded RNA dependent helicase (27). Since
many helicases have some SSB activity, it may nonspecifically
stimulate DNA polymerase e. If hsp60 is involved in either of
these processes, it would be expected to function in another
compartment besides the mitochondria. No function for hsp60
has been found in any other cellular compartment. However, two
recent studies have found evidence for hsp60 or hsp60-like
proteins in the cytoplasm. In one instance, hsp60 was found to
chemically crosslink with p21 ras in vivo (41). The authors
therefore propose a role for a small amount of a cytoplasmic form
of hsp60 in mammalian cells. A second finding is a family of
hsp60 homologues in 5. cerevisiae encoded by the TCP! genes
(42). We have investigated one possible non-mitochondiral role
of hsp60. Since GroEL, is a bacterial homolog of hsp60, is
induced in response to UV light (44), is involved in UV-induced
mutagenesis (45), and is involved in SOS repair (46), we tested
yeast mutants containing a temperature-sensitive hsp60 allele for
UV sensitivity. The mutants showed the same survival curves
as wild-type (data not shown). Therefore, if hsp60 is involved
in any of these processes, it is like one of the rare yeast genes
involved in DNA repair or mutagenesis that are not UV sensitive
(reviewed in 47 and 48). More experiments are necessary to
answer these questions.
ACKNOWLEDGEMENTS
We would like to thank Dr. Elizabeth Bertani for the chromosome
blot and Dr. Richard Hallberg for the unpublished temperaturesensitive mutants of hsp60.
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