Cell, Vol. 69, 1031-1042,

June

12. 1992, Copyright

0 1992 by Cell Press

SSL2, a Suppressor of a Stem-Loop Mutation in the HIS4 Leader Encodes the Yeast Homolog of Human EKG3 Keith D. Gulyas and Thomas Indiana University Department of Biology Bloomington, Indiana 47405

F. Donahue

Summary Reversion of haploid, Hisrl- yeast containing a stemloop mutation in the 5’ UTR that blocks HIS4 translation initiation identified four unlinked suppressor genes, SSLf-SSL4, which restore His4+ expression. The SSLP gene encodes an essential 95 kd protein with ATPdependent helicase motifs. SSL2 protein is 54% identical to the protein encoded by the human gene, ERCC-3, for which a defective form causes xeroderma pigmentosum and Cockayne’s syndrome. An SSLP allele made to resemble the defective ERCC.3 gene confers UV light hypersensitivity to yeast cells. Hence, SSLP is the functional homolog of ERCC3. However, the SSL2 suppressor gene does not restore HIS4 expression by removal of the stem-loop from DNA or the mRNA. We propose that SSL2 and ERCC-3 may have two functions, one defined by a UV repair defect, and a second essential function that is related to gene expression. Introduction Considerable controversy surrounds the initial events that lead to ribosomal binding of eukaryotic mRNA. According to the ribosomal scanning model, the ribosome binds near or at the 5’end of the message and then scans the leader region until it encounters the first AUG codon, the predominant signal for the start of translation (Kozak, 1980,198l). Although a number of molecular and biochemical experiments place the position of ribosomal binding within the first 15 nt of the mRNA(reviewed in Kozak, 1991) it remains unclear what facilitates the ability of the ribosome to bind the 5’ end of the message and what components of the preinitiation complex mediate this process. One step believed to be important for ribosomal binding to mRNA is the removal of secondary structure in the Y-untranslated region (5’ UTR) of mRNA (Sonenberg, 1988). The eukaryotic translation initiation factor 4F, elF-4F, has been implicated in this process. elF-4F is biochemically defined by three subunits (reviewed in Hershey, 1991): the 5’ capbinding protein (elF-4E), which has affinity for the posttranscriptionally modified 5’ end of eukaryotic mRNA, 7-methyl-G; the prototype DEAD box RNA helicase, elF4A; and ~220, a subunit of unknown function, which is cleaved to destroy elF-4F function during poliovirus infection (Etchison et al., 1982). It has been suggested (reviewed in Thach, 1992) that during the early events of translation initiation the cap-binding protein as part of elF4F binds the Send of the message. This would bring the

elF4A helicase to the S’end of mRNA, which would allow the unwinding of mRNA. Removal of secondary structure would then create a single-stranded region at the 5’ end of mRNA to provide a binding site for the ribosome. Although in vitro studies suggest that elF-4A in conjunction with elF4B can displace RNA-RNA hybrid molecules (Rozen et al., 1990), it remains unclear whetherthese proteins directly function in unwinding mRNA to allow ribosomal binding near the S’end of the message in vivo or whether the mechanism of ribosomal binding to mRNA might be more complex, perhaps requiring other components. Mutational studies at the HIS4 (Cigan et al., 1988; Donahue and Cigan, 1988) and CyC7 (Sherman and Stewart, 1981; Bairn and Sherman, 1988) genes in the simple eukaryotic yeast Saccharomyces cerevisiae demonstrate that the translation initiation process in yeast is consistent with fundamental features of the scanning model. The only mutations in the 5’ UTR of these genes that abolished expression were mutations of the AUG start codon and insertion mutations that introduced stable stem-loop structures in the leader. It has been demonstrated that the insertion of a stem-loop structure in the leader at HIS4 does not abolish expression by significantly changing the level of transcription or the position of the transcription initiation site (Cigan et al., 1988). This suggests that the HisC phenotype conferred to yeast strains by having a stem-loop structure in this region is a result of inhibiting HIS4 expression at a posttranscriptional level. Interpretations of similar insertion mutagenesis studies of mammalian genes (Pelletier and Sonenberg, 1985; Kozak, 1986) suggest that localized stem-loop structure in the 5’ noncoding region of mRNA blocks translation initiation either by preventing the ribosome from binding the 5’ end of the message or by preventing its migration toward the AUG start codon. In light of this, it seems likely that a genetic analysis of these types of mutations in yeast could provide a productive alternative approach in identifying components that promote ribosomal binding/scanning of mRNA. In this report, we describe a genetic reversion analysis of a his4 mutant allele that contains a stem-loop structure that presumably blocks the ability of the ribosome to bind or scan mRNA. Characterization of one of these exidentifies an essential tragenic suppressor loci, SSL2-7, protein with helicase signatures as important in overcoming the inhibitory effect of the stem-loop structure on HIS4 expression. Consistent with the selection scheme and the interpretation of the stem-loop mutation at HIS4, suppression appears to occur posttranscriptionally. Interestingly, SSL2 is the yeast homolog of the human gene ERCC-3, which has been found to be mutated in a patient with xeroderma pigmentosum (XP) and Cockayne’s syndrome (Weeda et al., 199Oa). An allele of SSLP that we constructed to resemble the defective gene in the XP patient also confers ultraviolet (UV) sensitivity. Our data suggest may have a nonessential function that SSLP and ERCC3 in nucleotide excision repair and an essential function in translation initiation.

Cell 1032

Figure 1. Selection Translation Initiation

SD-His

WI-

SUP

Results Genetic Identification of SSL Genes The method for identifying spontaneous mutations in unlinked genes that mediate ribosomal binding to mRNA is shown in Figure 1. The starting strain possesses the his4376 allele that contains a 36 bp insertion sequence with perfect dyad symmetry, %GGAAlTCCCGGATCCGGGCCCGGATCCGGGAATTCC-3’, in the DNA region that corresponds to the HIS4 leader (Cigan et al., 1966). This inserted sequence lies 10 nt downstream of the S’end of the HIS4 transcription initiation site and 50 nt upstream of the AUG that defines the start site of HIS4 translation (Donahue.et al., 1962; Nagawa and Fink, 1965). We predicted that when the his4-376 allele was transcribed in yeast cells, a thermodynamically stable stem-loop structure would exist in the 5’ UTR of the HIS4 message (Figure 1). Consistent with the notion that a highly stable secondary structure exists in the HIS4 message is the fact that strains containing the his4-376 allele have a His- phenotype on synthetic dextrose medium lacking histidine (SD-histidine) (Figure 1). Previous characterizations of the his4-376 transcript made in yeast demonstrated that insertion of the stem-loop structure in the 5’ UTR of HIS4 did not significantly alter the levels of HIS4 mRNA (Cigan et al., 1966). However, translation of HIS4 was drastically impaired by the insertion mutation being 1% of wild-type levels as measured in HIS4-IacZ fusion strains (Cigan et al., 1966). Haploid, His- yeast strains containing the his4-376 allele were reverted to His+ on SD-histidine plates (Figure 1). Revertants were genetically analyzed in crosses with a his4 deletion strain. Some revertants showed only a 2+: 2-segregation ratio for growth on SD-histidine, indicative of a reversion event at HIS4. Other His+ revertants when crossed to a his4 deletion strain each showed the meiotic ratios 2+:2-, 1+:3-, and 0+:4-, representative of a mutation in an unlinked gene that acts in trans to restore His4+ expression. Presumably, this latter classof revertantscontained mutations in genes that encode components of the translation initiation complex that can melt out or otherwise bypass the stem-loop structure to facilitate ribosomal

Scheme Factors

for

Identifying

The 43s preinitiation complex (left dumbbellshaped figure) is incapableof initiating (top part of figure) on the his4-376 message (center), which has a highly structured leader engineered into the gene in order to block ribosomal binding/scanning. This results in a His- phenotype on synthetic medium (SD-His) lacking histidine (top right). Selection for His+ revertants identified the SSLB7 suppressor mutant, which encodes a mutated transacting factor that allows the ribosome to initiate protein synthesis on the his4-316 message (bottom left and center) and results in a His+ phenotype (bottom right). The SC?+ wild-type strain JJ565 @VT) and SSLL-1 suppressor strain JJ566 (SUP) are shown after 2 days incubation on synthetic dextrose medium lacking histidine.

binding/scanning of the HIS4 mRNA. Further genetic characterization of these strains (T. F. D. and K. D. G., unpublished data) has identified four distinct suppressor genes, SSL7-SSL4 (SSL: Suppressor of Stem-Loop), which when mutated restore HIS4 expression despite the presence of the stem-loop structure mutation in the 5’UTR of the message. SSL7 suppressor strains are temperature sensitive (Ts-) for growth on enriched medium, and the Ts- phenotype is recessive and cosegregates with the suppressor phenotype in genetic crosses. SSL7 is weakly codominant, while SSLP, SSL3, and SSL4 are dominant suppressors. Isolation of the SSLP Wild-Type Gene As part of our genetic characterization of SSL suppressor strains, we noticed that haploid strains containing both the SSL7-7 and SSLP-7 suppressor genes maintained a Tsphenotype for growth on enriched medium but were His-, that is, no longer exhibiting asuppressor phenotype (Table 1)-an indication of a functional interaction between the two gene products. However, the His+ suppressor phenotype could be restored by a simple complementation test. Mating the SSL7-7, SSLP-7 haploid strain to an SSL7+, SSL2+ haploid strain of the opposite mating type conferred a His+ phenotype upon the resulting diploid strain (Table 1). The His+ phenotype was also restored when the double suppressor mutant was mated to either an SSL7-7 or an SSLP-7 haploid strain (Table 1). Hence, the presence of either an SSL7 or an SSLP wild-type gene in the genetic background of the double suppressor mutant strain appeared to be sufficient to restore the His+ suppressor phenotype. In addition, each of the latter diploid strains was phenotypically distinguishable from the other. SSL7-71 SSL7-7, SSLP-7I+ diploids are Ts- for growth on enriched medium owing to the homozygous nature of the SSL7-7 suppressor allele in this strain. In contrast, SSL7-7/+, SSLP-7ISSLP-7 diploids are Ts+, as the Ts- phenotype conferred by the SSL7 suppressor allele is recessive to the domin ,“t SSLl+ allele. These observations served as the basis cor cloning the SSLP wild-type gene. The wild-type SSL2 gene was cloned from a YCp50 genomic bank based on its ability to restore a His+ phenotype to the SSL7-7, SSLB7 haploid suppressor strain. As pre-

SSLP, the Yeast 1033

Homolog

of Human

ERCC-3

the plasmid, the SSL7-7 suppressor gene being present in single copy and conferring a recessive Ts- phenotype (Table 1). Plasmids were isolated from seven His+, Ts- transformants that were suspected to contain the wild-type SSLP gene on the plasmid. Plasmids from three of these transformants contained a common 4.6 kb Hindlll fragment in the cloned segment of DNA. This fragment when subcloned into YCp50 was able to restore the His+ suppressor phenotype to a haploid SSLl-1, SSLP-7 mutant, as expected if the intact SSLP’ gene was present on the 4.6 kb Hindlll DNA fragment. Genetic analysis of yeast strains transformed with an integrating Ylp5 plasmid containing a 5 kb EcoRl DNA fragment, which overlaps most of the 4.6 kb Hindlll fragment, demonstrated tight linkage of the URA3’ plasmid to the SSLP locus. These observations provided the genetic evidence that the cloned DNA we identified was derived from the SSLP chromosomal region. Characterization of the SSLP Wild-Type and Suppressor Genes The DNA sequence of the cloned region identified an 843 aa open reading frame (ORF) that begins with an ATG and is capable of encoding a protein with a calculated M, value of 95,280 (Figure 2). Primer extension analysis of total

The 3.9 kb DNA region encompassing the SSLP ORF is shown. Both strands of the DNA were sequenced in their entirety either on single-stranded phage DNAor double-stranded plasmid DNA. Downward pointing arrows indicate potential transcription start sites as determined by primer extension of total RNA from a wild-type (SSL2+) yeast strain. The SSLP-7 suppressor mutation is indicated by an asterisk beside tryptophan-427 (w); the mutation is a transversion of the second base of the codon from TGG to n-G, replacing tryptophan ON) with leucine (L). Sequence motifs common to helicase-like proteins are underlined and numbered with roman numerals. Motifs are designated in the manner of Hodgman (1988) and Burgess et al. (1990). The Ndel site used to construct the SSL2-XP allele is indicated with an open, upward pointing arrow. An SSLPDEAD allele was constructed to change codons 490 and 491 from GF (valine, V) to GCT (alanine, A) and CAT (histidine, H) to SAT (aspartate, D), respectively (see Experimental Procedures). Both new codons have a high bias (Sharp et al., 1986) for codons in abundantly expressed genes in yeast. These mutations introduce an additional Alul site in the gene. The homology to the human ERCC-3 gene begins at amino acid 59 (methionine, M) and extends to the end of the coding sequence. As suggested by the primer extension data, there may be alternative transcripts of SSLP mRNA. The two potentially shorter transcripts would be expected to use the methionine (M) at amino acid position 19.

Cdl 1034

SSL2 ERCC-3

?56I..*DSDHQVQP

Figure 3. Comparison and ERCC-3

of the Predicted

Protein

Sequences

of SSLP

The single letter amino acid sequence of the SSLP and ERCC-3 genes are aligned. SSL2 appears above ERCC3 (Weeda et al., 1990a). The SSL2 sequence comparison begins at amino acid position 59 (Figure 2). Boxed regions indicate shared amino acid identity. In total, the SSL2 and ERCC-3 gene products are 54% identical, with few gaps needed to be introduced in the alignment. The sequences were first aligned with the GCG program Bestfit (Genetics Computer Group, Inc., Madison, WI) and then aligned by eye.

RNA from a wild-type strain (data not shown) indicates that the region is transcribed and reveals three major transcription start sites at nucleotide positions -31, +16, and +22 relative to the ATG that defines the 643 aa ORF (Figure 2). The identical DNA region was isolated from an SSLP-7 suppressor strain by the integration/excision method (Winston et al., 1963). DNA sequence analysis identified a point mutation, which maps at nucleotide position 1996 (Figure 2), that changed the 427th codon of the ORF from tryptophan (TGG) to leucine (TTG). Gene disruption of one copy of the 843 aa ORF with &her URA3’ or TRPl’ in a wildtype diploid strain resulted in two viable and two inviable meiotic products upon sporulation, with all viable spores being either Ura- or Trp-, respectively. LEU2+, CEN plasmids containing either the SSLP wild-type or suppressor gene are capable of rescuing the lethal effect of an SSL2 gene disruption in haploid strains. Furthermore, attempts to make these disruption strains lose the SSLP gene on a

plasmid were unsuccessful, suggesting that SSLP is also essential for vegetative growth of yeast cells. Based on these studies we conclude that the 843 aa coding region encodes the SSLP gene product, which has an essential function in yeast. This is consistent with the design of our selection scheme to identify genes that encode components of the translation initiation complex that function in ribosomal binding/scanning of mRNA. Inspection of the amino acid sequence of SSLP indicated a number of sequence motifs (Figure 2) characteristic of helicaselnucleic acid binding proteins (Hodgman, 1988; Gorbalenya et al., 1988, 1989; Linder et al., 1989; Company et al., 1991). Two motifs, motif I which begins at amino acid position 389 (GAGKT) and motif II which begins at position 487 (LDEVH), define a putative nucleotide binding fold (Rossmann et al., 1974; Walker et al., 1982). The mutation found in the SSLP-7 suppressor mutation alters an amino acid that maps between these two motifs (Figure 2). Interestingly, an allele of PRP76, which was isolated as a suppressor of a splice junction mutation in yeast, has also been characterized to have a mutation that altered an amino acid that maps between motifs I and II of the nucleotide binding fold (Burgess et al., 1990). In addition, mutations that alter the function of the elF-4A helicase protein have also been defined to map between motifs I and II (Schmid and Linder, 1991). The presence of these sequence motifs in the SSLP coding region and the position of the SSLP-7 mutation between motifs I and II suggest that the SSL2 protein might function as a helicaselnucleic acid binding protein in yeast. A mutation in a gene product of this type might be expected to alter its function to suppress the inhibitory effect of a stem-loop structure positioned in the HIS4 leader region. SSLP Is the Yeast Homolog of the Human Gene ERCC-3 Upon searching the GenBank data base, we discovered SSLP to be the yeast homolog of the human gene ERCC-3, displaying 54% amino acid sequence identity (Figure 3). When conservative amino acids are taken into consideration, the sequence similarity is upward of 80%. This high degree of sequence similarity extends throughout the protein and includes the above mentioned motifs observed in helicaselnucleic acid binding proteins. However, the two proteins appear to differ in length; the SSLP protein is predicted to be 58 aa longer than the ERCC-3 protein at the NH?-terminal end (Figures 2 and 3). ERCC-3 was identified as a human genomic clone that complemented the UV-sensitive defect associated with a mutant rodent cell line (Weeda et al., 1990b). A mutation in ERCC-3 is also correlated with the human disease XP type B and Cockayne’s syndrome (Weeda et al., 199Oa). Both conditions are manifested by the inability of cells to efficiently repair UV-damaged DNA. Given this association, it was speculated that ERCC-3 was a putative DNA helicase involved in DNA repair (Weeda et al., 1990a). OtherxGidence supporting this proposal included limited amino acid sequence homologies to DNA-binding proteins and a predicted nuclear targeting signal in the ERCC-3 sequence. These sequences are also conserved in the

SSLP, the Yeast 1035

Homolog

of Human

ERCC-3

Dose (J/m*) Figure

4. UV Survival

Profile

Saturated yeast cultures of strains KG99 (SSLZ, boxes), JJ586 (SSLP7, triangles), KG106 (SSL2-DEAD, circles), and KG1 19 (SSL2XP, crosses) were diluted in water and plated onto YEPD. Cells were immediately irradiated under a germicidal lamp for various times (Song et al., 1990; Haynes and Kunz, 1981). Plates were then incubated at room temperature in the dark for at least 24 hr and shifted to 30%. Colonies were ing fraction (see Experimental Procedures) was determined. The ordinate represents log surviving fraction, while the abscissa displays the dosage of UV irradiation in Joules per square meter.

SSLP coding region (data not shown). The only detectable version of ERCC-3 expressed in the XP patient contained a mutation in a splice acceptor site of an intron located in the carboxyl terminus of the gene. This mutation causes improper splicing of the primary ERCC-3 transcript that results in a frameshift mutation in the carboxyl end of the ERCC-3 coding region (Weeda et al., 199Oa). As a result of this frameshift mutation, the ERCC-3 protein is predicted to be synthesized without the last 43 carboxy-terminal amino acids in the ERCC-3 coding region. A Carboxy-Terminal Deletion of SSLP Renders Yeast Hypersensitive to UV Light To investigate the possibility that the SSLP gene product is the functional homolog’of the ERCC-3 product, we constructed altered allelic forms of SSLP that could substitute for the wild-type SSLP gene and tested these strains for hypersensitivity to UV light. One allele was constructed in an attempt to produce an Ssl2 protein in yeast that mimicked the truncated form of the ERCC-3 protein predicted to be made in the XP patient. The SSLP gene contains a single Ndel restriction site at nucleotide position 2965 (Figure 2). Through this unique Ndel site we were able to construct an SSLP allele, SSLP-XP, that would encode an Ssl2 protein that is missing 94 aa from the C-terminal end. The second allele we constructed mutated motif II of the putative nucleotide binding fold from LDE&l to the cannonical LDEAD. We wished to alter this motif in such a way as to perturb the predicted “ATP” binding/hydrolysis function of this domain in Ssl2 but not to the extent of incompatibility with the essential function of the SSLP gene.

Strains carrying the SSLBXP or SSLP-DMD genes were constructed by plasmid shuffle, replacing a plasmid-borne copy of the wild-type SSL2 gene in a TRP1::SSLP gene disruption background. These strains as well as SSLP wild-type and suppressor strains were grown in liquid YEPD medium, diluted, and plated out on YEPD plates. Sets of plates were then irradiated with increasing doses of UV light and incubated in the dark for 24 hr in order to eliminate activation of the yeast photo-induced repair pathway (Haynes and Kunz, 1981). The number of surviving cells, as indicated by their ability to grow into a colony, was determined and compared with the UV sensitivity profile of the isogenic strain that contained the wild-type SSLP alleleon aplasmid. Asshown in Figure4, the UVsensitivity profiles for strains containing the SSL2-1 and SSLS-DEAD genes are indistinguishable from the strain that contains the wild-type SSLP gene. In contrast, the strain containing the SSLP-XP gene as its sole copy of SSLP is hypersensitive to UV irradiation; lo4 times more cells were killed at doses of 15 J/m’ or greater in comparison with the other strains (Figure 4). This effect cannot be attributed to some adverse effect this allele might have on the growth properties of yeast, as the SSL2-XP strain grows as well as the SSLP-7 strain and better than the SSL2-DEADstrain (Table 2); neither of the latter two strains displays any hypersensitivity to UV irradiation (Figure 4). In light of the sequence identity (Figure 3) and the fact that the truncated forms of SSLP and ERCC-3 proteins produce similar phenotypes for UV sensitivity in their respective organisms, we conclude that the SSLP and ERCC-3 gene products perform similar functions in yeast and humans, respectively. SSL2 Suppression Is Not Related to DNA Repair One simple interpretation of our analysis is that the SSLP gene product functions in the yeast DNA repair process, perhaps being a DNA helicase, as was proposed for the function of the ERCC-3 gene product (Weeda et al., 1990a). It therefore stands to reason that the mechanism by which the SSL2-7 allele suppresses the stem-loop structure mutation at HIS4 might be related to DNA repair. One possibility is that as a result of a mutation in the SSLP gene, the Ssl2 suppressor protein removes (repairs) the stem-loop from the DNA to produce a HIS4 allele void of such a structure. The end result is that there would no longer exist an obstacle to HIS4 expression, and therefore, a His+ phenotype is restored. One prediction of this mechanism is that removal of the stem-loop structure from the DNA would result in a stable H/S4+ phenotype that is genetically linked to the HIS4 locus and no longer dependent on

Table 2. Phenotype

of Strains

Carrying

Different

SSL2 Alleles

Strain

SSLP Allele

Doubling Time (min)

his4-376 Suppression

UV Resistance

KG99 JJ586 KG106 KG1 19

SSLP SSLP-1 SSL2-DEAD SSL2-XP

03 96 102 93

+ ND

+ + +

ND, not determined.

Cell 1036

Table 3. Segregation

SUP

of His Phenotype 2+:2-

Expected Ratios Repair of HIS4 Suppression in trans Observed Ratios MATa SSLP1 his4-316 MATa SSL2 his4A401 MATa SSLP his4A401 MATa SSLB1 his4-316

CTAG 1+:3-

WT CTAG

WT

SUP

0+:4-

6

0

0

1

4

1

1

20

6

4

22

4

1

the SSLP suppressor gene. This is easily detected in a genetic cross to a his4 deletion strain, as we would expect a 2+:2- segregation ratio among meiotic products indicative of the His+ phenotype being linked to HIS4 (Table 3). In contrast, if SSLP suppressed his4 by a mechanism that did not involve generating a change in the HIS4 DNA, then we would expect 0+:4-, 1+:3-, and 2+:2- segregation ratios on SD-histidine in a ratio of 1:4:1 (Table 3). These latter segregation ratios are indicative of the SSLP suppressor gene being unlinked to HIS4 and its gene product required to be always present in a cell, acting in trans to confer a His+ phenotype. Although our initial genetic analysis of the SSLP suppressor strain was inconsistent with a mechanism of suppression that was related to DNA repair, we tested this possibility in a more direct fashion. A haploid SSLP suppressor strain was first streaked out on SD-histidine medium to isolate purified single His+ colonies. If the mechanism of SSLP-7 suppression relates to DNA repair, then maintenance of selective growth conditions would provide ample opportunity for SSLP-7 to “repair” the stem-loop structure at HIS4 in the entire population of these cells by generating a new HIS4’ allele in each cell. These His+ strains were then crossed to a his4 deletion. As shown in Table 3, tetrad analysis of this cross shows that the His phenotype segregates with meiotic ratios of 2+:2-, 1+:3-, and 0+:4- in the expected proportions, 1:4:1. Hence, the is not being repaired or stem-loop structure at his4-376 altered to arrive at a His+ phenotype. To dispel any doubt as to the action of the suppressor on the DNA, the his4-316 alleles contained in the SSLP suppressor strains that had been maintained on SD-histidine plates were isolated, and the corresponding leader region was sequenced. The DNA sequence of the HIS4 leader region indicated that the suppressor strains contained the stem-loop insertion sequence, which is identical in sequence to the his4 region on a plasmid (Figure 5) that was used originally to conallele (Cigan et al., 1988). We conclude struct the his4-376 that the mechanism of SSLP suppression is not related to DNA repair. SSLP Suppression Does Not Remove the Stem-Loop from the HIS4 mRNA In contrast to removal of the stem-loop structure from DNA, an alternative mechanism for SSLP suppression might be to generate a HIS4 message that no longer pos-

Figure

5. his4-316

Molecular

Characterization

Lanes marked C, T, A, and G are sequencing reactions using doublestranded plasmid DNA. The sequencing lanes on the left (SUP) were generated from the his4-376 allele isolated from an SSLP-7 suppressor strain that had been maintained on SD-histidine medium. The sequencing lanes on the right (WT) are derived from the plasmid used to construct the hid-376 allele, as previously described (Cigan et al., 1988). The sequence of the insertion in the his4-376 is displayed to the left of the sequencing lanes, indicating the symmetry of this segment of DNA. Compressions in some regions prevent full resolution of the inserted sequence, but are identical in both the WT and SUP sequences. The two lanes on the far right are the primer extension products of the his4-316 transcript made from the isogenic parent (JJ565) and suppressor (JJ586) strains. Total RNA (10 Kg) from JJ565 was used as template in lane WT, while total RNA (IO ug) from JJ586 was used in lane SUP. The primer and procedure is described in the Experimental Procedures and Cigan et al. (1988). Below the autoradiograph is a diagramatic representation of the primer extension reaction, in which an antisense -P-labeled oligonucleotide, indicated by a thicker solid line at the right end, is annealed to the HI.94 coding sequence. Reverse transcriptase extends the oligonucleotide up to the point of the insertion (I), which is refractive to its passage. The products of the reaction map the presence and position of the stem-loop, but do not identify the 5’ end of the mRNA (compare with Cigan et al., 1988).

sesses the stem-loop structure mutation. One possibility is that SSLPencodes a transcriptional factor and as a result of a mutation has an altered specificity for the HIS4 transcription initiation site. The end result might be that transcription initiation at HIS4 would now occur 3’ to the stem-loop structure, generating HIS4 mRNA lacking the stem-loop structure. If the position of this new transcription initiation site is still 5’ to the AUG start codon of the HIS4 coding region, then a His+ phenotype would be achieved. Alternatively, the Ssl2 protein could have RNA splicic@excision function and it could be that as a result of amxation, it now hasaffinityforthestem-loopstructure in HIS4 mRNA and removes this sequence posttranscriptionally to arrive at a His+ phenotype. To investigate these possible mechanisms of suppres-

SSLP, the Yeast 1037

Homolog

of Human

ERCC-3

sion we isolated total RNA that was made from the SSLP wild-type parent and the SSLP-7 suppressor strains, both of which contained the stem-loop mutation at the HIS4 locus, and analyzed the structure of the 5’ region of the HIS4 transcript by primer extension. As shown in Figure 5, primer extension analysis of the HIS4 transcript made from a His-, SSLP wild-type strain identifies a termination position near the base of the insertion mutation. This does not reflect a transcription initiation site at HIS4, which has been well documented to map at position -60 relative to the AUG start codon (Donahue et al., 1982; Nagawa and Fink, 1985). Instead, this reflects the termination position of the primer extension reaction as a result of a stable and localized stem-loop structure being present in the HIS4 mRNA, as previously demonstrated (Cigan et al., 1988). The identical termination position in HIS4 mRNA is also observed when primer extension analysis is performed with RNA isolated from an SSLP-7 suppressor strain (Figure 5). Overexposure of our primer extension gels does not identify any unique HIS4 transcript in the SSL2-7 strain that might start 3’ to the site of the insertion mutation. These data suggest that an alternative transcription initiation event is not occurring at HIS4 as a result of the SSLP suppression event, nor is the stem-loop being removed from the mRNA. The levels of HIS4 mRNA identified from total RNA in an SSL2+ and an SSLP suppressor strain do appear to be different (Figure 5, compare lanes WT and SUP). In independent experiments, we observe approximate 2- to 3-fold increases in his4-316 transcripts in SSLP-7 versus SSL2+ strains. However, 2- to 3-fold increases in his4-376 transcriptional levels cannot be solely responsible for suppression, as assays of His4 8galactosidase levels produced in his4-316-/acZ fusion strains indicate that the level of HIS4 expression in an SSLP-7 suppressor strain is increased approximately 7.5-fold. In light of our analysis of the stem-loop mutation in his4 DNA and RNA, we suggest that the mechanism of SSLP suppression is related to a posttranscriptional process, presumably at the level of translation initiation. Discussion Using a genetic reversion scheme at the HIS4 locus in yeast, we have attempted to identify by mutation components of the translation initiation complex that are important for ribosomal binding/scanning of mRNA. Previous mutational studies in our laboratory of the HIS4 leader region indicated that no simple sequence could be altered to inhibit ribosomal binding/scanning of the HIS4 message (Cigan et al., 1988; Donahue and Cigar& 1988). Therefore, we have relied on the introduction of a stem-loop structure in the HIS4 leader to provide a physical barrier to the early steps of translation initiation. In light of the general assumption that unwinding of secondary structure from mRNA is considered to be an important first step toward ribosomal binding of mRNA (Sonenberg, 1988; Thach, 1992), it stands to reason that mutant initiation factors would confer altered specifities to the translation initiation complex to melt out or bypass the stem-loop mutation and promote ribosomal binding/scanning of the mRNA.

Characterization of the SSLP suppressor strain identified a gene with properties that are in agreement with our selection strategy. SSLP encodes a protein with characteristicsof RNA bindinglhelicase proteins that would be anticipated to “unwind” secondary structure to promote ribosomal binding/scanning of mRNA. The position of the mutation in the SSLP suppressor gene is similar to the position of mutations that alter the function of the helicaselike proteins encoded by the elF-4A (Schmid and Linder, 1991) and PRPl6 (Burgess et al., 1990) genes; the elF-4A protein has been shown to possess ATP-dependent RNARNA duplex displacement activity in conjunction with elF-4B (Rozen et al., 1990) and the Prpl6 protein has been shown to possess RNA-dependent ATPase activity (Schwer and Guthrie, 1991). In addition, our primer extension analysis suggests that an alteration of the start site of HIS4 transcription or excision of the stem-loop from his4 is not the mechanism of SSLP suppression (Figure 5) but rather that the mechanism involves translating the h&4-376 mRNA despite the presence of the stem-loop structure in the 5’ UTR. Although our primer extension analysis is consistent with this interpretation, it does not eliminate other possible mechanisms for arriving at the suppressor phenotype. However, we are confident that the mechanism is not related to DNA repair and therefore that Ssl2 must have an additional function. The primer extension analysis indicates that the level of his4-376 transcript increases in the SSLP-7 strain in comparison with an SSLP wild-type strain (Figure 5, compare lanes WT and SUP). This could suggest that Ssl2 is an essential protein that suppresses his4-376 by increasing transcript levels. In independent experiments we see an approximate 2- to 3-fold increase in the level of his4-376 transcript in an SSLP-7 strain when compared with an SSLP wild-type strain. However, based on previous characterizations of transcriptional expression of other types of his4 translation initiation mutants and suppressor strains (Valavicius et al., 1990) and the 1% level of His4 expression observed from a his4-376-/acZ fusion strain in an SSL2+ genetic background (Cigan et al., 1988) an approximate 2- to 3-fold increase in transcript levels is considered to be insufficient to arrive at a His+ phenotype. In fact, we and others have observed increases in HIS4 transcript levels as indirect effects of translational suppression. The positive activator of HIS4 transcription, GCN4, is subject to translational control (Hinnebusch, 1990). Other suppressors we have isolated that encode translation initiation factors (Donahue et al., 1988; Cigan et al., 1989) have been shown, in addition to their translational suppressor activity, to elevate HIS4 transcript levels 3- to 4-fold by altering GCN4 translational control (Williams et al., 1989; Valavicius et al., 1990). Also, enhanced translation of the his4-376 mRNA might be expected to enhance the stability of this mRNA, as has been observed for the effects of suppression of a his4 frameshift mutant (Leeds et al., 1991). Other mechanisms that could potentially give rise to suppression, such as modification of the stem-loop RNA sequence (Bass and Weintraub, 1988) or conceivably overcoming a potential inhibition of transport of HIS4 message

Cell 1038

from the nucleus by the stem-loop, have not been ruled out. Nevertheless, in light of the design of the selection scheme and our characterization of SSLP suppression, we favor the interpretation that SSLP plays an essential role during the translation initiation process in yeast. Other observations we have made may support this interpretation. Conditional suppressor alleles of SSL7 have in vivo polysome defects at the restrictive temperature, indicative of a mutation in a protein that functions during translation initiation (H. Yoon, E. Pabich, and T. F. D., unpublished data). Furthermore, conditional SSL7 suppressor strains have thermolabile defects in the cell-free, in vitro translation system (S. Miller and T. F. D., unpublished data). The fact that haploid, SSL7-7, SSLP-7 double mutants lose the His+ suppressor phenotype (Table 1) could indicate that both essential proteins interact and function in the translation initiation step. One surprising outcome of our analysis is that SSLP is the functional homolog of the human gene ERCC-3, which has been implicated to be involved in the autosomal recessive human disease, XP. It has been assumed that ERCC-3 is a putative DNA helicase based on limited sequence similarities to DNA-binding proteins (Weeda et al., 1990a), its ability to complement a UV repair defect associated with a mutant rodent cell line (Weeda et al., 1990b), and the fact that a mutant form of the gene exists in a patient with XP and clinical symptoms of Cockayne’s syndrome (Weeda et al., 199Oa). The fact that a mutation in SSLP, made to mimic the clinically characterized mutated form of ERCC-3, results in UV-sensitive yeast cells would appear to further point to the importance of this protein in DNA repair. However, it is clear from our analysis that Ssl2 and presumably the human homolog, ERCC-3, do not solely function in DNA repair. First, the SSLP-7 suppressor protein does not alter the his4-376 DNA to arrive at a His+ phenotype or confer a UV-sensitive defect. Second, the SSLP-DEAD mutation confers poorer growth properties upon yeast cells than SSLP-XP does, but the former strain does not show increased sensitivity to UV light (Figure 4 and Table 2). One interpretation from our studies is that Ssl2 and ERCC-3 have dual functions. One function is related to DNA repair, as defined by the fact that a carboxy-terminal deletion mutation in either SSLP or ERCC-3 confers a UV repair defect in yeast and mammals, respectively. The second function is defined by our SSLP suppressor, which affords expression of HIS4 despite the presence of a stemloop structure mutation in the 5’UTR. Ssl2 and by extrapolation, ERCC3, may both interact with mRNA and DNA. Dual functions for a protein involved in DNA repair have been suggested (Friedberg, 1988). The RAD3 gene was identified as a mutant that was defective in DNA repair in yeast and encodes a gene product with characteristics of a “helicase” protein (Naumovski et al., 1985; Reynolds et al., 1985). The human gene ERCC-2 encodes a RAD3 homolog and is also implicated to be involved in DNA repair (Weber, et al., 1988,199O). Rad3 has been proposed to have an essential function in addition to its function in nucleotide excision repair (Friedberg, 1988). In addition, Rad3 protein has ATP-dependent RNA-DNA duplex dis-

placement activity, although the biological role of this activity has not been established (Bailly et al., 1991). There appears to be an interesting parallel between studies of Ssl2 and Rad3. Both genes are essential and may have a nonessential function in DNA repair, and both encode helicase-like proteins with mammalian functional homologs. Therefore, our studies and the observations on RADB may be establishing a precedent that helicase-like proteins may be capable of interacting with both DNA and RNA to perform different biological functions. However, our analysis could be interpreted differently, by speculating that Ssl2 and ERCC3 perform a single and essential function during translation initiation. In this interpretation, each gene product would be important for the translational expression of a group of genes, among which one or more is related to DNA repair or is a gene that controls the expression of a DNA repair enzyme. Similar types of mutations in each corresponding protein might then lead to the same effect, namely, poor expression of a gene that has an adverse effect on the DNA repair pathway. This would suggest that yeast as well as mammalian cells might share a similar mechanism for controlling expression of a DNA repair enzyme(s) at the level of translation initiation. Interestingly, recent studies have now established that the mechanism of controlling translation initiation in mammalian cells via phosphorylation of elF-2 by elF-2 kinases is conserved in yeast (Dever et al., 1992). By analogy, the repair defect observed by mutating either SSLP or ERCC-3 might be an indirect effect of perturbing a common translational control mechanism. It is important to note that mutations in the RADGgene of yeast confer UV sensitivitity, and this gene encodes one of the many ubiquitin-conjugating enzymes in yeast (Jentsch et al., 1987). How mutations in thisgene alter DNA repair has not been established. However, Rad6 serves as a good example of a gene product that might not be expected to interact directly with DNA, even though mutations in this gene confer a DNA repair defect. Based on our selection scheme, we suggest that if Ssl2 proves to play a role in translation initiation it will be to promote ribosomal binding to mRNA or scanning toward the AUG. Ssl2 does not correspond to any of the previously described translation initiation factors in eukaryotic cells (Hershey, 1991). We actually anticipated isolating suppressor forms of the putative helicase elF-4A that might have improved “unwinding” activity to implicate a biological role for this protein in unwinding secondary structure from mRNA. Although Ssl2 has some sequence identity to elF-4A, it clearly is not yeast elF-4A, as both elF-4A genes have been cloned from yeast (Linder and Slonimski, 1989) and neither is the SSLP gene. Perhaps other SSL suppressor genes we have identified will turn out to be suppressor forms of elF-4A. Nevertheless, our ability to identify SSL2 as a suppressor of stem-loop structure mutations could suggest that elFr4A may not be the only “helicase” protein involved in the early steps of translation initiation. Given the rasrof helicase-like molecules that have been identified (Chang et al., 1990; Wassarman and Steitz, 1991) and the numerous helicase proteins involved in intron splicing steps (Ruby and Abelson, 1991) one might expect that

SSLP, the Yeast 1039

Homolog

of Human

ERCC-3

an RNA interactive process such as translation initiation might have its fair share of helicase-like proteins to promote ribosomal binding/scanning. Experiments are currently underway to establish the mechanism of SSLP suppression and its physiological relevance. Experimental

Procedures

Yeast Strains and Genetic Methods All strains used in this analysis are related to TD28 (MATa ura3-52 inol-73). an ascospore derivative of yeast strain S288C (MATa) that has been used extensively for the characterization of HIS4 transcription (Donahue et al., 1982, 1983; Nagawa and Fink, 1985) and translation initiation (Donahue and Cigan, 1988; Cigan et al., 1988). Standard genetic techniques and media used for these studies have been described (Sherman et al., 1972). The construction of yeast strains containing the his4-376secondary structure allele (previously referred to as -51/-5082) has been described (Cigan et al., 1988). The His4- parent strain JJ565 (MATa ura3-52 inol-73 his4-376) was used to select for spontaneous His+ revertants by replica plating lawns grown on YEPD (yeast extract, peptone, dextrose) plates to SD-histidine plates. His+ revertants were characterized genetically for complementation ability, dominance and recessiveness, and Mendelian segregation (T. F. D. and K. D. G., unpublished data). Pertinent to the reported studies, the SSL suppressor strains JJ588 (SSLL-7) and JJ836 (SSL7-7) were characterized in genetic crosses with the his4 deletion strain 44-l B (MATa leu2-3, -112 his4-407). The double suppressor mutant strain 802-7A (MATa ura3-52 inol-73 his4-376 SSL7-7 SSLB7) that was used to clone the wild-type SSL2 gene (Table 1) is an ascospore derived from a cross between strainsJJ586and5559C(MATa/eu2-3, -772inol-73his4-376SSLl-7). The diploid strains TFDlOO, TFDlOl, and TFDlOP (Table 1) were constructed by mating strain 802-7A to yeast strains JJ565, JJ586, and JJ636, respectively. An in-frame hi&-316-&Z fusion gene as part of YCp50 was used to transform the isogenic strains JJ565 (SSL2+) and JJ586 (SSLP7) to Ura’. The construction of this plasmid was previously described (Cigan et al., 1988) and contains all 5’ noncoding sequences that are necessary for the transcription and regulation of HIS4 expression. Strains were grown in SD medium lacking uracil, and extracts were prepared and assayed for P-galactosidase activities as previously described (Donahue and Cigan, 1988). Isolation and Characterization of the SSL2 Wild-Type and Suppressor Alleles The wild-type SSLP gene was cloned from a YCp50 genomic clone bank (Rose et al., 1987). SSL2+-containing clones were identified on the basis of restoration of the His+ suppressor phenotype while maintaining the Ts phenotype of the double mutant SSL7-7, SSLB7 strain, 802-7A, conferred by the SSL7-7 allele. The strategy for cloning SSL2 is presented in Table 1 and explained in the text. Plasmids were isolated from the His+, Ts transformants EP1062, EP1066, and EP1072, and restriction analysis identified a 4.6 kb Hindlll and 5 kb EcoRl DNA fragment that was common to the insert of all three plasmids, two of which appeared to have identical inserts. Both DNA fragments were isolated from the two plasmids with different inserts and subcloned into YCp50, Ylp5, and pBR322. The YCp50 plasmids containing these subcloned DNA fragments were used to transform yeast strain 802-7A, and transformants were tested for the ability of each subcloned DNA fragment to restore a His+ phenotype. The 5 kb EcoRl fragment in Ylp5 (plasmid ~1084) was used to transform yeast strain JJ559 (MATS ura3-52 inol-73 his4-316) to Ura+. A Ura’ transformant was then crossed to yeast strain Gl-5C (MATa ura3-52 leu2-3, -712 his4-376 SSL2-7) and subjected to tetrad analysis. The Ura’ and the His+ suppressor phenotype each segregated 2+:2-. Ura’ meiotic products were always His-, and Ura- meiotic products were always His+, indicative of the Ura3’ phenotype associated with Ylp5 being tightly linked to the SSL2+ gene as a result of integration of ~1084. The URA3’ gene was used to disrupt the SSLP gene (Rothstein, 1983) that was present as a 4.1 kb Hindlll-EcoRI fragment in a pBR322 vector. A 1.4 kb Clal fragment within the SSLP coding region (nucleotide positions 1339 to 2702, Figure 2) was deleted and replaced with

an Ahall-Clal fragment from YEp24 that carries the intact URA3 gene. The disrupted SSLP gene was isolated as a Bglll-EcoRI fragment of DNA, which contained contiguous SSL2 sequences flanking SSL2:: URA3+ and was used to transform the diploid yeast strain, EKP84 (MA TaMA Ta ura3-52/ura3-52 his4-306lhis4-306 leu2-3, -7 12/leu2-3, -772), to Ura’ to generate yeast strain KG68. For some experiments an SSLP::TRPI+ gene disruption strain was used. This strain was constructed by cloning the entire TRPl gene on a 1.25 kb Pvull fragment from plasmid pJJ246 (Jones and Prakash, 1990) into the same SSLP Clal sites used for the Ura3’disruption. except that they were first filled in with Klenow to generate blunt ends (Maniatis et al., 1982). An Sspl DNA fragment containing SSL2::TRPI’was then used to transform the diploid strain KG83 (MATaIMATa ade2/+ ade5/+ his7-2ihis?-2 leu2-3, -7 72//eu2-3, -7 72 tfpl-289/trp7-289 ura3-52/ura3-73) to Trp’ to generate yeast strain KG86. Tetrad analysis of the gene disruption strains KG68 and KG86 resulted in 2 viable:2 inviable meiotic products. The SSLB7 allele was isolated by the integration/excision method (Winston et al., 1983). A 2.2 kb Hindlll fragment that is contiguous with the 4.6 kb Hindlll that contains the SSL2+ gene was sucloned into the Hindlll site of the yeast plasmid Ylp5. This plasmid, ~1105, was restricted at a unique Bglll site in the 2.2 kb Hindlll insert and used to transform the SSLP-1 suppressor strain, JJ586, and the wild-type strain, JJ565, to Ura’. Genomic DNAwas digested with BamHI, ligated, and used to transform Escherichia coli to ampicillin resistance. Restriction with BamHl results in the isolation of the downstream chromosomal copy of the SSLP gene. The plasmids were isolated, and the DNA was confirmed by restriction analysis to contain the 4.6 kb Hindlll fragment that contains the corresponding SSL2 gene. The 4.6 kb Hindlll DNA fragment containing either the SSL2 or SSLB7 alleles were subcloned into a Hindlll site of the LEU2-C.&V4 vector pSB32 and used to transform yeast strain KG83 to LeuS+. Tetrad analysis indicated that the presence of either SSLP or SSLBf as part of this vector resulted in two, three, or four viable meiotic products, indicative of the ability of either SSL2 gene to rescue the lethal effect of the SSL2::TRP gene disruption in haploid meiotic products. To determine whether the SSLP gene was essential for vegetative growth of yeast cells, Leu+, Trp+ haploid products were grown nonselectively in YEPD liquid medium, plated out for single colonies on YEPD medium, and replica plated to SD-leucine-tryptophan plates. Over 1000 colonies were analyzed, and all were Leu+ and Trp’, indicative of the inability of the SSLP::TRP7+ strain to lose the sole copy of SSLP on the plasmid.

DNA Sequence Analysis Sequencing reactions were carried out by the method of Sanger (1977). The 4.6 kb Hindlll fragment derived from the plasmid identified in the transformant EP1062 served as the source of the wild-type sequence. The SSLP wild-type gene contained on this 4.6 kb Hindlll fragment was restricted at a unique Pvull site to generate 1.9 kb Hindlll-Pvull and 2.7 kb Pvull-Hindlil DNA fragments. The 1.9 kb fragment was cloned into the Hindlll and Hincll sites of M13mpll and mp18 vectors to give both orientations. The 2.7 kb fragment was cloned into the same sites of mpl 1. The 2.7 kb clone was further digested with EcoRI, which cleaves 0.5 kb off the Hindlll-proximal side of the fragment. This 2.65 kb Pvull-EcoRI DNA fragment was cloned into the EcoRl and Hincll sites of mpll, giving the opposite orientation relative to the 2.7 kb Pvull-Hindlll mpll construction. Initial sequences were generated with a universal primer. Subsequent sequences were generated with synthetic (17-mer) oligonucleotides synthesized to be complementary with sequences determined in the previous round of sequencing. Oligonucleotides were obtained from the Institute for Molecular and Cellular Biology (Bloomington, IN). The DNA sequence across the Pvull junction was accomplished using double-stranded plasmid DNA as the template. Confirmation of part of the SSLT sequence was determined from a Narl-Sphl DNA fragment subcloned into the Accl and Sphl polylinker of M13mp19 and a Blgll-Narl DNA fragment subcloned into a M13mplO. The entire SSL2+ sequence (Fig ure 2) was determined for both strands of the DNA. The SSLB7 mutation was identified by double-stranded sequencing. The SSLP-7 allele was isolated on a plasmid from the suppressor strain, JJ586, as described above. Confirmation of the mutation in the SSLB7 suppressor gene was determined by sequencing the same region in the SSLT

Cell 1040

gene that was isolated from the parent strain, JJ565, by the integration/ excision method as described above. The his4316 allele was isolated from SSLB7 strain JJ586 by the integration/excision method using a modification of the method previously described for isolating other his4 alleles (Donahue et al., 1963). Yeast strain JJ586 was first streaked out on SD-histidine in order to isolate single colonies. Plasmid B113, which contains the upstream region of HIS4 as part of a 3.0 kb EcoRl DNA fragment (Donahue et al., 1962) was restricted with Bglll and used to transform three strains that were derived as His+ colonies. Chromosomal DNA was isolated, digested with Hindlll, and ligated. Plasmid DNA was isolated in E. coli and restricted with BamHl and EcoRI, two restriction sites that are within the stem-loop and therefore diagnostic of the stem-loop insertion mutation in the his4-376 leader region. Plasmids from all three of these strains were sequenced in the region of the stem-loop insertion using an oligonucleotide, 5’~CATCAATTAACGGTAGAATCGG-3, that is complementary to positions +lO to +31 in the HIS4 coding sequence as previously described (Cigan et al., 1988). Sequences were compared with the DNA sequence of the same region located in plasmid ~627 (formerly referred to as p-51/-50B2), which was used originally to construct the his4376 allele (Cigan et al., 1988). Construction of SSLZ-XP and SSLP-DEAD Strains The SSLBXP allele was constructed using plasmid ~1359-1, which contains SSL2 on a Hindlll-EcoRI fragment ligated in pBR322. This plasmid was first cut with Ndel, and this site was filled in with Klenow and dNTPs. The DNA was then digested with Hindlll, and the DNA fragment corresponding to the upstream portion of SSL2 (Figure 2) was recovered from agarose. This DNA fragment was cloned into the Clal and Hindlll restriction sites of YCp50, the Clal site having been initially filled in with Klenow to make a blunt end that was compatible with ligation to the blunt-ended Ndel site in the SSL2 DNA fragment. The SSLBDEAD allele was constructed using the oligonucleotidedirected in vitro mutagenesis system version 2 (Amersham). The single-stranded template was the M13mpll that contained the 2.7 kb Hindlll-Pvull fragment as described above. The oligonucleotide (5’~CTTGACGAAGCTGATGTGGTGCC-3’) used as primer in the poly merization reaction has two mismatches that alter amino acid codon 490 from GTT to GCT and amino acid codon 491 from CAT to GAT in the SSLP coding region (Figure 2). The oligonucleotide was also designed to introduce a new Alul restriction site as part of the two mismatches. Thus, candidates for successful mutagenesis were first screened for an additional Alul restriction site, followed by DNA sequencing. The SSLP coding region containing both mutations as part of a 2.5 kb Apal-EcoRI DNA fragment was isolated from the phage and substituted for the same fragment in the SSL2 wild-type gene in plasmid ~1359-1. The intact SSLP-DEAD gene was then subcloned from this plasmid into YCp50 as a 4.5 kb Hindlll-EcoRI DNA fragment. The SSL2-XP and SSLIDEAD genes in YCp50 were introduced into yeast strains as the sole source of the SSLP gene by plasmid shuffling (Schatz et al., 1988). Yeast strain KG93 was constructed by transforming diploid KG86 with plasmid ~1533, which carries the SSLP wild-type gene on a L&/2-CfN4 plasmid. KG93 was induced to undergo meiosis. A haploid ascospore, KG99, that was Trp’, Leu’, and Ura- was then transformed to Ura’ with the SSL2-XP or SSLBDEAD YCp50 plasmid. Leu’, Ura+ transformants indicative of strains containing either the SSLBXP or SSLBDEAD genes and the SSLP wildtype gene were grown nonselectively in YEPD liquid medium and then plated on YEPD plates. Trp’, Ura’, Leu- colonies were indicative of haploid strains that have lost the wild-type SSL2 gene as part of the LEUP+, CEN4 vector but maintained either the SSL2-XPor SSLBDEAD gene as part of YCp50, respectively. UV Sensitivity Assay Yeast strains KG99 (SSLT), JJ586 (SSLB7), KG106 (SSLZ-DEAD), and KG119 (SSL2-XP) were grown in liquid YEPD medium, serially diluted, and plated onto YEPD plates. Plates were immediately exposed to increasing doses of UV radiation (Fink and Styles, 1974) then incubated at room temperature in the dark for at least 24 hr, and transferred to 30% UV irradiation doses were calculated using the Model UVX digital radiometer (UVP, Inc.). Surviving colonies were counted, and the number was multiplied by the dilution factor and divided by the starting cell population. The log of this fraction was then

plotted against the UV dose to generate Figure 4. Each data point is the average of three plates per UV dosage from the same experiment, Primer Extension The 5’ mapping of the his4-376 transcripts (Figure 5) was determined by primer extension using [@*P]ATP 5’-end labeled oligonucleotide and AMV-reverse transcriptase as previously described (Cigan et al., 1988). Total RNA was isolated from the isogenic yeast strains JJ565 (SSLT) and JJ586 (SSLP-7), which were grown in SD complete medium to an ODa of 1.6 to 1.7. Primer extension analysis was performed using the same oligonucleotide described above for DNA sequencing of the his4-376 allele, which was also used previously to characterize the hi&376 transcript as well as the transcript made in other stemloop insertion mutants of the HIS4 leader region (Cigan et al., 1988). The HIS4 wild-type transcript has previously been determined by both Sl nuclease and primer extension methods (Donahue et al., 1982; Nagawa and Fink, 1985) to be located 60 nt 5’of the AUG start codon. Acknowledgments We thank members of our laboratory for helpful discussions, Mike Myers and Ed Pabich for cloning the SSL2 wild-type gene, Julie Johanson for the initial genetic isolation and characterization of suppressor strains, Mimi Zolan for helpful advice on the UV repair assays, and Peter Cherbas, HsiuJung Lo, Barry Polisky, and Mimi Zolan for helpful suggestions on the manuscript. This work was supported by Public Health Service grant GM32263 from the National Institutes of Health awarded to T. F. D., and K. D. G. is a predoctoral trainee on the NIH genetics training grant GM07757. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

5, 1992; revised

April 2, 1992.

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Accession

The accession M94176.

number

Number for the sequence

reported

in the article

is