GENETIC ANALYSIS OF STAPHYLOCOCCAL NUCLEASE ...The three-dimensional structure of staphylococcal...

$: GENETIC ANALYSIS OF STAPHYLOCOCCAL NUCLEASE ...The three-dimensional structure of staphylococcal nuclepe has been solved by X-ray diffraction analysis and recently refined to 1.5 A$
Copyright 0 1985 by the Genetics Society of America

GENETIC ANALYSIS OF STAPHYLOCOCCAL NUCLEASE: IDENTIFICATION OF THREE INTRAGENIC “GLOBAL”

SUPPRESSORS OF NUCLEASE-MINUS MUTATIONS

DAVID SHORTLE’ AND BETH LIN

Department of Microbiology, State University of New York, Stony Brook, New York I 1 794

Manuscript received January 8, 1985 Revised copy accepted April 10, 1985

ABSTRACT

A collection of 77 unique missense mutations distributed across the gene encoding staphylococcal nuclease (nuc) has been assembled. These mutations were induced by random gap misrepair mutagenesis of the cloned gene and were identified in E. coli transformants expressing reduced levels of nuclease activity. Four nuc- mutations which alter amino acid residues at positions outside of the active site region of the enzyme were submitted to a second round of mutagenesis, and characterization of several independent NUC+ isolates lead to the identification of three second-site suppressor mutations within the protein-coding sequence of the nuc gene. On separation from the mutation orig- inally suppressed and recombination with a number of other nuc- mutations, all three suppressors displayed the property of “global” suppression, i.e., phenotypic suppression of the nuclease-minus character of multiple different alleles. A simple and generally applicable strategy was used to obtain efficient homologous recombination between plasmids for purposes of mapping nuc- mutations, mapping second-site suppressors and constructing double mutant combinations from pairs of single’ mutations.

HE detailed characterization of mutations induced by random mutagenesis T and identified by their mutant phenotype has in the past provided an invaluable means of initiating study of a wide variety of complex biological phenomena, prominent examples being the regulation of gene expression in prokaryotes, the morphogenesis of bacteriophage and the biosynthetic path- ways of several natural products. One complex biochemical phenomenon to which the potential of this approach has yet to be fully explored is the very general problem of how the primary amino acid sequence of a protein precisely specifies the folding pathway to, and the final stability of, its native conformation. Since the amino acid sequence information that determines the folding/stability of a protein is encoded by a segment of DNA, the practical problem of extracting folding/stability information from a particular gene should, in principle, be analogous to other genetically definable problems. In other words, the systematic characterization of gene mutations that alter folding/ stability may permit the identification of those residues that specify critical

’ Present address: Department of Biological Chemistry, T h e Johns Hopkins University School of Medicine, Baltimore, Maryland 21 205.

Genetics 110 539-555 August, 1985.

540 D. SHORTLE AND B. LIN

steps in the folding pathway or essential noncovalent bonds in stabilizing the native conformation and may help elucidate the general patterns through which the information distributed across the polypeptide chain is integrated to determine the final structure.

Since the phenotypic characterization of mutations that alter protein folding/ stability will demand extensive quantitation of the properties of purified mutant proteins, a model system chosen for genetic analysis ideally should encode a relatively stable protein that is easy to isolate. In addition, since physical methods for analyzing folding and stability require reversible denaturation and renaturation of the protein (KIM and BALDWIN 1982; PFEIL 1981), model proteins should be amenable to this type of analysis without the common but serious complication of aggregration on unfolding. And finally, it is important to note that the extraction of meaningful kinetic and thermodynamic information from denaturation/renaturation studies is significantly more straight- forward for those proteins that behave as single folding domains and do not oligomerize at high protein concentrations (PFEIL 198 1).

Staphylococcal nuclease is one of a relatively small number of proteins that satisfies the above criteria. This enzyme, which is also referred to as micrococ- cal nuclease, has been extensively characterized over the past 20 y r by many investigators (reviewed by ANFINSEN 1973; TUCKER, HAZEN and COTTON 1978, 1979a,b,c). A single polypeptide chain, 149 amino acids in length, this mono- meric protein lacks disulfide bridges and reversibly folds and unfolds as a single domain at physiologic pH and high protein concentrations (ANFINSEN, SCHECH- TER and TANIUCHI 1972). Structurally, it does not require metal ions or pros- thetic groups, although one calcium ion is a cofactor for enzymatic activity. The three-dimensional structure of staphylococcal nuclepe has been solved by X-ray diffraction analysis and recently refined to 1.5 A resolution (COTTON, HAZEN and LECC 1979). One compact globular domain built of both a-helices and p sheets, staph nuclease is classified as an a + @-protein (LEVITT and CHOTHIA 1976; RICHARDSON 198 1).

T o apply the genetic approach to the specific problem of folding/stability of staphylococcal nuclease, the gene encoding this protein was cloned from the Foggi strain of Staphylococcus aureus, and a plasmid-based genetic system utiliz- ing Escherichia coli as the host cell was developed (SHORTLE 1983). Because this gene is expressed at a level in E. coEi sufficient to produce detectable amounts of active enzyme, a simple replica plate assay can be employed as a phenotypic screen for nuc- mutations, nucf recombinants and NUC+ revertants. Although by no means a phenotype specific for mutations that alter folding or stability, it was anticipated that large perturbations in structure or stability might lower the specific activity of mutant enzymes or might lead to proteolysis with concomitant reduction in the amount of nuclease accumulated in E. cola colonies. Therefore, a large collection of nuc- mutations might include some that would prove, on biochemical characterization, to encode proteins with altered folding/stability.

In this report, the isolation, genetic mapping and nucleotide sequence analysis of a number of missense mutations are described that were detected with

GENETIC ANALYSIS OF STAPH NUCLEASE 54 1

this phenotypic screen. In addition, reversion analysis of several nuc- alleles led to the identification of three intragenic suppressors which are able to suppress the mutant phenotype of as many as 15 different mutant alleles.

MATERIALS AND METHODS

Materials: Oligonucleotides 5’-GCCAAGCCTTGACG-3 ‘, 5’-TGCACTTGCTTCAGGA-3’ and 5’-GGACGTGGCTTAGC-3’ were custom synthesized by P-L Biochemicals and used in dideoxy sequencing reactions to prime within the nuc gene from codon 105 (antisense), codon 55 (antisense) and codon 90 (sense), respectively. T h e S , diastereomers of thymidine 5’-0-( 1-thiotriphosphate), 2’-deoxyadenosine 5‘-0-( 1-thiotriphosphate), 2’-deoxyguanosine 5’-0-( I-thiotriphosphate) and 2‘- deoxycytosine 5’-0-(l-thiotriphosphate) were gifts from S. J. BENKOVIC and F. ECKSTEIN. All enzymes were purchased from New England BioLabs, except M. luteus DNA polymerase I was obtained from Miles Laboratories. A molecular model of staphylococcal nuclease (LabQuip type) built by JOHN MACK (66 Overlook Terrace 8B, New York, New York 10040) from the refined coordinates (Brookhaven Protein Data Bank) was used to draw conclusions about the positions of amino acid residues in the native conformation of the wild-type protein.

Bacterial strains and plates: Endonuclease 1-minus E. coli strains 1100 (endAI thi-l supE44), MM294 (endA1 thi-1 hsdRI7 supE44) and DHI (endAl thi-1 hsdRI7 recA1 gyr96) were obtained from the E. coli Genetic Stock Center at Yale University. All transformants with plasmid DNA (DAGERT and EHRLICH 1979) were selected on LB agar plates (MILLER 1972) containing 200 pg/ ml of ampicillin and were replica plated using clean velvetine squares to toluidine blue-0 DNA (TBD) agar plates (LACHICA, GENICEORCIS and HOEPRICH 1971; SHORTLE 1983). Nuclease activity was scored after incubation of TBD plates at 37” for 8-16 hr.

Recombinant DNA techniques: Small amounts of plasmid DNA were prepared from E. coli transformants by a modification of the method of BIRNBoIM-and DOLY (1979). Approximately 1 pg of mutant plasmid DNA was cleaved with Clal and the linear DNA dissolved in a small volume after phenol extraction and ethanol precipitation. This ClaI-cleaved DNA was used for genetic mapping, as template for DNA sequencing and for construction of double mutants. Heteroduplexes between two linear plasmid DNAs were formed by first mixing 50 ng of each DNA in a final volume of 8 pl of 0.1 N NaOH/O.I mM EDTA and incubating at 37” for 1 5 min. After addition of 10 PI of 0.08 N HCI/O.l M Tris.HCI, pH 8.0, renaturation was promoted by incubation at 50” for 1 hr. Ten microliters of this solution was then immediately used to transform 100 pl of competent E. coli cells.

Cap misrepair mutagenesis: Mutagenesis was made as random as possible by introducing one random target site for nucleotide misincorporation per molecule. First, a single nick was induced by treating covalently closed supercoiled molecules with DNase 1 plus ethidium bromide as previously described (SHORTLE and BOTSTEIN 1983). T h e position of the 3’ terminus was further randomized by converting the nick into a short single-stranded gap using exonuclease 111 at 30” in 20 mM Tris.HCI, pH 8.0/7 mM MgC12/7 mM 2-mercaptoethanol/lOO p g / d of heat-treated bovine serum albumin with an amount of enzyme sufficient to convert 95% of nicked molecules in 15 min into a form resistant to covalent closure by T4 DNA ligase (SHORTLE and BOTSTEIN 1983). After two phenol extractions and an ethanol precipitation step, 250 ng of this gapped plasmid DNA was subjected to gap misrepair mutagenesis with a-thiophosphate nucleotides (SHOR- TLE et al. 1982). Reaction 1 of this protocol was modified by reducing the concentration of MnC& from 0.2 to 0.1 mM and increasing the a-thiophosphate nucleotide concentration from 0.1 to 0.25 mM. This change in reaction conditions should eliminate drift of the 3’ terminus prior to misincorporation by blocking the 3’ to 5’ exonuclease of the Klenow fragment polymerase. (Inhibition of this exonuclease appears to reduce a previously observed phenomenon of mutational hot spots at the first position downstream of sites where the a-thiophosphate nucleotide could be correctly incorporated.) Modifications in reaction 2 of this protocol consisted of reducing the temperature from 26” to O ” , increasing the magnesium acetate from 1 to 5 mM and reducing the MnCb from 2 to 1 mM-all in an effort to reduce the frequency of frameshift mutations due to strand slippage.

DNA sequencing reactions: T h e Sanger method of DNA sequencing (SANGER, NICKLEN and COUL-


SON 1977) was applied directly to denatured linear plasmid DNA (SMITH et al. 1979) using three synthetic oligonucleotides to prime synthesis from defined sites within the nuc gene.

RESULTS AND DISCUSSION

A general strategy f o r directing homologous recombination between plasmids: Once a collection of phenotypic mutations has been isolated after random mutagenesis, their genetic characterization usually proceeds by first mapping each mutation to a defined genetic interval, often by methods involving homologous recombination between DNA molecules. At subsequent stages in genetic analysis, double mutants generated by recombining pairs of single mutations may be constructed to search for specific interactions between genetic loci. AI- though homologous recombination is an extensively utilized tool for mapping mutations and for constructing combinations of mutations in some genetic systems, it has not played an important role in genetic analyses carried out on genes cloned into the standard E. coli plasmid vectors such as pBR322 for two reasons. (1 ) It is technically difficult to introduce and maintain two closely related, and, therefore, incompatible, plasmids in the same E . coli cell. (2) T h e spontaneous rate of homologous recombination is relatively low, especially for nearby markers, so that many colonies must be searched in order to recover a recombinant plasmid. Although restriction enzymes plus DNA ligase can sometimes provide an in vitro nonhomologous pathway for catalyzing recom- binational exchanges, a simpler and more general means of directing homologous recombination between E . coli plasmids carrying mutant alleles of a cloned gene would facilitate the genetic characterization of phenotypic mutants.

In Figure 1 a strategy is outlined that has permitted the efficient recovery of recombinants between pBR322-derived plasmids carrying different types of mutations in the cloned gene for staphylococcal nuclease. To direct recombination between two homologous plasmids A and B by this strategy, each plasmid is first cleaved at a different unique restriction site, with one site (plasmid B) always involving a linker oligonucleotide inserted at the position of a defined deletion of DNA sequences (termed a linker-bracketed deletion). When equi- molar amounts of the two linear molecules are denatured and reannealed, approximately 50% of the product will be renatured linear homoduplexes and 50% will be circular heteroduplexes with a single-stranded gap covering those DNA sequences that are deleted in plasmid B. On transformation of this mixture of DNA species into E. coli, the linear molecules will give rise to very few transformants, whereas the circular heteroduplexes will efficiently transform (SHORTLE 1983). Presumably, the nonhomologous ends created by the linker sequences are efficiently excised, and the missing segment of the strand derived from plasmid B is repaired by the cell by copying the information from the intact strand derived from plasmid A. T h e initially gapped DNA strand will become a genetic recombinant if parental information from the intact strand is copied onto it with no erasure of its parental information. In other words, a genetic recombinant is generated whenever the single-stranded gap of a

GENETIC ANALYSIS OF STAPH NUCLEASE

Restriction

A

Restriction Enzyme 1

4 4 MIX, DENATURE. REANNEAL

543

,wild-type

-mutant

B

,mutant

A ---mutant __*__

- D 1 2 ,mutant1+2 4 2 4- E - , mutant 1 1 1

CIRCULAR HETERODUPLEXES WITH DELETION GAPS

FIGURE 1 .-A, Outline of steps for directing homologous recombination between two plasmids by formation of heteroduplex molecules containing a deletion gap. See text for a detailed discussion. In B, C and D the configuration of mutations relative to the deletion gap are shown for genetic crosses to map a minus mutation (B), to map a second-site suppressor mutation (C) and to construct a double mutant allele from pairs of single mutations (D). Note that only those outcomes of in vivo gap repair are shown that do not eliminate the information at the site of the mismatch, indicated by the X.

heteroduplex molecule is filled in without concomitant repair of the mutant/ wild-type mismatch flanking the single-stranded gap.

As shown in Figure lB, when a heteroduplex is formed between a point mutation on plasmid A and a simple linker-bracketed deletion on plasmid B, wild-type recombinants can potentially be formed if the mutation falls outside the deletion interval, but not if the mutation falls under the deletion interval. In a matrix of crosses involving linker insertion mutations in the nuc gene heteroduplexed against linker-bracketed deletions 60-350 nucleotides in length, it was demonstrated that approximately 50% of transformants scored as nut+ recombinants whenever the mutation fell outside the deletion interval (SHORTLE 1983). Presumably, in the remaining 50% of transformants that do not give rise to wild-type recombinants, repair of the single-stranded gap results in erasure of the information in the gapped DNA strand that represents the wild-type counterpart of the mutant site. As described below, this same high frequency of recombinants was found in all crosses involving single base substitution mutations falling more than five nucleotides outside of a given deletion interval.

Two additional applications of recombination via deletion gap heteroduplexes used in the genetic analyses presented in this report are outlined in


Figure 1C and D. Second-site suppressor mutations can be mapped by crossing a mutant-suppressor pair with a set of deletions, all members of which include the initial nuc- mutation. The map interval of the suppressor is then defined as the increment in the deletion interval over which the initial nuc- mutation can no longer be separated from the suppressor by recombination. In addition, combinations of different mutations can be constructed by first introducing into one mutant plasmid a linker-bracketed deletion with an interval that in- cludes the mutation in the other plasmid.

In the crosses described below, a simple replica plate test was used to facilitate scoring of transformants for NUC+ or NUC- recombinants. Even though few foreign genes cloned on E. coli plasmids have such an easily scoreable mutant phenotype, this strategy of recombination should be generally workable because recombinants appear at a very high frequency in those crosses that can give rise to recombinants, and, therefore, only a small number of progeny (i.e., 10-20) need to be analyzed to score the result of a mapping cross or to construct a double mutant.

Isolation, mapping and sequence analysis of nuclease-minus mutants: To isolate a collection of mutant alleles of the gene encoding staphylococcal nuclease by random mutagenesis and phenotypic screening, a genetic system based on a modified pBR322 plasmid carrying the cloned nuc gene from S. aureus was first developed (SHORTLE 1983). This 4. l-kb plasmid, referred to as pFOG30 1, can be transformed into E . coli by selection for ampicillin resistance and scored for the production of active staph nuclease by replica plating transformant colonies to agar plates containing toluidine blue-0 and high molecular weight DNA (TBD plates). As many as 300 colonies/plate can be scored for nuclease activity by comparing the size of the blue-stained patch of transferred bacterial cells to the diameter of the surrounding pink halo formed as the outwardly diffusing nuclease hydrolyzes the DNA in the plate (LACHICA, GENICEORGIS and HOEPRICH 1971).

The method of gap misrepair mutagenesis with a-thiophosphate nucleotides (SHORTLE et al. 1982) was applied to purified pFOG301 DNA in vitro. Initial results with this method had suggested that it could potentially induce most, and possibly all, of the 12 types of base substitution mutations and, therefore, make available mutant pools with as many as one-third of all possible single amino acid substitutions represented. T o make the mutagenesis as random as possible, a single target site was induced per molecule by first nicking covalently closed pFOG3O 1 with pancreatic DNase1 plus ethidium bromide. Since the nuclease gene constitutes approximately 12% of the sequences in this plasmid, one of eight molecules should receive a nick at one more or less random site in the nuc gene. To further randomize the target site for mutagenesis, which with this method is the first nucleotide position downstream of the 3’ hydroxy terminus of a single-stranded gap, the nick was converted into a short gap by limited reaction with the 3’ to 5’ activity of exonuclease 111.

In separate experiments, each of the four a-thiophosphate nucleoside tri- phosphates was misincorporated using the randomly gapped pFOG3O 1 DNA as substrate. To obtain pure clones of mutant molecules, the mutagenized

GENETIC ANALYSIS OF STAPH NUCLEASE 545

SIGNAL PEPTIDE (?) NUCLEASE A UAA

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 J 1 I I I \ I l I 1 1 I l l I I I l l 1

-80 -60 -40 -20 - I *I +20 *40 *60 +80 400 *I20 *I40 A 13

A IO

12 i . . 1 12 23 1 21 16 1 14 I 13

FIGURE 2.-The set of BamHI linker-bracketed deletion derivatives of plasmid pFOG3O 1 used in the genetic crosses described in this report. The construction of deletions d e l l to de16 has been described previously (SHORTLE 1983). Deletion plasmids d e l l 0 to de114 were assembled by the same strategy using other plasmids with unique EamHI linkers inserted at an XmnI site (codon -9), an AvaII site (codon +55) , a unique Hind111 site (codon +101) and a unique Nael site (75 bp downstream of the stop codon). The construction of de151, de152 and de153 is described in the text. Each of these three deletion plasmids carries a suppressor mutation at the site indicated by an X. The numbers at the bottom indicate the total number of mapped nuc- mutations falling under each deletion interval.

DNA was transformed into the strain MM294, and plasmid DNA prepared from a pool of 3000-10,000 transformants was used in a second transformation of strain DH 1. In each of these experiments, approximately 5000 transformants were scored for nuclease activity, with the result that 0.5-1.5% of colonies displayed either a reduced level or no nuclease activity (NUC-). If the gap misrepair reactions had gone to completion and thus yielded a misincorporated nucleotide at one site on one strand in every molecule, the maximum frequency of mutations expected to fall within the nuc gene would be one-half of 12% or approximately 6%. If it is assumed that the efficiency of induction of base substitutions is close to this maximal value (SHORTLE et al. 1982), the observed frequencies of 0.5-1.5% NUC- colonies suggest that the phenotypic assay used to identify mutant alleles can detect a significant fraction of all point mutations.

After purifying several micrograms of plasmid DNA from each mutant isolate and cleaving an aliquot at the unique ClaI site located outside of the gene (SHORTLE 1983), the nuc- mutation was genetically mapped by crossing with the BamHI linker-bracketed deletions d e l l , de12, de13, del4, de15 and d e l l l . Of 127 nuc- mutations, 126 could be positioned to a single unique map interval, and the distribution of mutant sites is shown at the bottom of Figure 2. In all


crosses except two, wild-type recombinants either were not detected (<0.5%) or they constituted approximately half of all transformants (35-60%).

Once the DNA sequence was determined for a particular mutation, the position of that mutation relative to the ends of the single-stranded gap formed in heteroduplex crosses could be inferred. The two crosses that gave significantly fewer than 50% recombinants (nucG145 X de12 gave 4% wild type and nucA121 X de15 gave 20%) both involved a wild-type mutant mismatch separated by 3 bp from one end of the single-stranded gap. In one cross with a mismatch located 5 bp away (nucG126 x de14) and two crosses with mismatches 6 bp away from the gap (nucAIl3 X de12; nucC213 X de14), the frequency of recombinants was in each case 50%. Consequently, the genetic map interval defined operationally in this type of heteroduplex cross is only 3-4 bp wider than the physical deletion interval (SHORTLE 1983).

T o identify the specific nucleotide sequence change in each mutant, the DNA sequence of the entire physical interval corresponding to the mutation’s map position was determined using the Sanger chain termination method (SAN-

GER, NICKLEN and COULSON 1977). In addition to providing a collection of defined mutant alleles specifying variant forms of staphylococcal nuclease that could be submitted to subsequent physical chemical analysis, the nucleotide sequence also helps to define the spectrum of mutations induced by gap misrepair mutagenesis with a-thiophosphate nucleotides.

Among the 104 mutations sequenced, 98 unique mutations were identified, 77 of which were single base substitution missense mutations and are listed in Table 1. Of the remaining 21 mutations, ten resulted in nonsense codons, six were multiple noncontiguous base substitution and five were single nucleotide insertions. The number of single base substitutions recovered after mutagenesis with a-thiodATP were G to A (two), T to A (eight) and C to A (two), whereas after mutagenesis with a-thiodTTP they were A to T (12), G to T (seven) and C to T (nine). These data confirm the earlier finding that gap misrepair mutagenesis with these two nucleotides can induce each of the possible types of base substitutions (SHORTLE et al. 1982). The base substitutions recovered after mutagenesis with a-thiodCTP were A to C (15), T to C (two) and G to C (two), whereas after mutagenesis with a-thiodGTP, only A to G (13) and T to G (seven) substitutions were recovered. From these data it appears that, with the current reaction protocols, the G and C nucleotides are being misincorporated efficiently opposite some, but perhaps not all, noncomplementary nucleotides in the template strand.

Since a high resolution X-ray diffraction map has been determined for staphylococcal nuclease with a competitive inhibitor bound in the active site (COT- TON, HAZEN and LEGG 1979) and a number of amino acid residues have already been assigned roles in catalysis (reviewed by TUCKER, HAZEN and COT- TON 1979b), each missense allele can be tentatively classified with regard to the mechanism behind its nuclease-minus phenotype based on the position of the altered amino acid residue in the native conformation. For example, mutations that change residues known to bind the calcium ion cofactor or the 5’ phosphate of pdTp (the presumed site of phosphodiester bond hydrolysis)


TABLE I

Missense mutations in the staphylococcal nuclease gene (nuc)

Amino acid Enryme Presumed Amino acid Enzyme Presumed substitution" Allele no.' activity' defectd substitution" Allele no! activity' defectd

547

L7F K9T I15N 118M D19V D21E D21E D21Y T22P V23G V23F Y27S T33S F34Y F34Y R35G R35I L37V L37F L37S L38S V39G D40G D40G D40E T41P E43G E43K H46R H46Y K49N E52V E52K A60V K63N A69T E73A V74F E75G E75D

C209 C207 T207 c 2 1 0 A108 C205 T 2 2 1 T216 G145 G120 AI 13 G I 4 9 C218 A105 T205 G109 A1 14 c 2 0 2 C226 C223 G I 0 3 C216 GI 15 c 2 1 1 C213 GI26 G I 5 3 B203 G117 B204 A127 A106 T210 T228 c 2 2 2 T217 G129 T235 G102 C214

++ ++ + ++ 0 0 0 0 0 0

++ + ++ + + 0 0

+/- 0 0

++ 0 0 0

++ 0 0 0 + + + +

++ 0

++ ++ + 0

+/-

+/-

Ind Ind Ind Ind Cat Cat Cat Cat Ind Ind Ind Ind Ind Ind Ind Cat Cat Ind Ind Ind Ind Ind Cat Cat Cat Cat Cat Cat Bind Bind Bind Bind Bind Ind Ind Ind Ind Ind Ind Ind

E75K F76S F76V F76Y D77A D77G G79S G79D D83G D83V K84E R87G R87C G88D L89F A90S Y91D Y91S I92N D95E D95A M98I

N 1 OOY ElOlG ElOlA L103V L103F V104D R105H L108V NI 18D H121P E122K K127Q E129K K133T L137S N 138D W 140G W 140C

T213 G119 G131 T236 G116 G125 A121 T202 GI28 T204 GI11 c201 T212 T203 T232 T211 G121 c 2 2 1 T215 C225 C227 T222 T224 GI52 C103 C104 C224 T225 A126 C204 GI06 C206 T227 C229 T233 C219 G114 G I 4 4 C203 T206

+ 0

++ 0 + + 0 + 0 0 + 0

++ 0 0 0 + 0 0 0

++ ++ ++ 0 0 + 0

0

0

++ + + 0 0

+/-

+/-

+/-

+/-

+/-

+/-

Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind

Bind Cat Cat Ind

Bind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind Ind

"The first letter designates the wild-type amino acid in the one letter code and is followed by

'The letter preceding the allele number designates the a-thiophosphate nucleotide used to

"Nuclease activity was scored as wild type (4+), moderate (++), weak (+), very weak (+/-) or

dAlteration of a catalytic residue (Cat), a presumed binding residue (Bind) or a residue that

the residue position and the amino acid substitution encoded by the mutation.

induce the mutation.

zero (0) by visual comparison of halo sizes formed on TBD agar plates.

indirectly effects activity (Ind).


probably lead to defects in catalysis, whereas mutations that alter residues along a cleft extending out from the catalytic site are classified as potentially defective in polynucleotide binding. Approximately one-third of the nuc- mutations listed in Table 1 can be assigned to one of these two classes. The remaining two-thirds, however, change residues which are outside of the active site/cleft region. Presumably, these mutations confer a nuclease-minus phenotype through one or more mechanisms that do not directly involve binding and hydrolysis of substrate. Examples of defects at the level of mutant protein product that could result in loss of nuclease activity include failure of transport into the periplasmic space, aggregation, proteolysis, distortion of the active site or gross unfolding to an enzymatically inactive species. Alternatively, but less likely, defects occurring at the level of mRNA accumulation or translation could result in a NUC- phenotype through diminished synthesis of an otherwise normal enzyme.

Reversion analysis : Before beginning the necessary biochemical characterization of the mutant nucleases made from specific nuc- alleles to definitively identify the mechanisms behind their nuclease-minus phenotypes, an attempt was made to study several of the mutants in the third or “indirect” class by reversion analysis (HARTMAN and ROTH 1973; JARVIK and BOTSTEIN 1975). T o search for intragenic suppressors of the five alleles nucGl31, nucA121, nucGl2S, nucGlO6 and nucA112, purified plasmid DNA carrying each mutation was subjected to random gap misrepair mutagenesis with the a-thiophosphate nucleotides dATP and dGTP. Mutagenized DNA was transformed directly into MM294 cells, and approximately 5000-20,000 transformants were screened for NUC+ colonies by replica plating to TBD agar. In each screen, from one to five positive colonies were identified, representing a reversion rate induced by this type of random mutagenesis of 1/1-4000 molecules. All but three of the 15 total isolates scored as significantly weaker in nuclease activity than wild type, suggesting they were not true revertants or accidental contam- inants of wild-type pFOGSO 1.

T o map the site of reversion, a small preparation of plasmid DNA from each isolate was cleaved with ClaI and then heteroduplexed with a set of linearized linker-bracketed deletions as outlined in Figure 1 C. After transformation with the reannealed mixture of DNAs, transformant colonies were replica plated to TBD agar and nuc- recombinants were scored. To reduce the background of spurious NUC- colonies arising from uncleaved deletion plasmids, revertant plasmid DNA was prepared form a EcoK modification-plus strain (MM294), the mapping deletion plasmids were prepared from an EcoK modification-minus strain (HB 10 1) and the heteroduplexes were transformed into an EcoK restriction-plus strain (1 100). As a result, virtually all uncleaved deletion plasmids fail to transform, whereas heteroduplexes with one modified strand transform with normal efficiency. Each revertant isolate was first heteroduplexed to the smallest deletion covering the nuc- mutation reverted and, if nuc- recombinants were recovered (indicating a second-site suppressor was present), progressively larger deletions with intervals including the original nuc- mutation were used. The results of these crosses are shown in Table 2.


TABLE 2

NUC+ revertants isolated a f e r random mutagenesis

549

Results of crosses with deletions

Isolate No Reverting nuc- allele no." NUC- recombinants recombinants Site of revertant substitution

GI31 GI de14, 12, 13 de114 Second H 124R (F76V) A1 de14 Same V76F

A2 de14, 12, 13 de114 Second H124L A3 de14, 12, 13 de114 Second H124L

A121 G1 de14 Same S79G (G79S) G 2 de14, 1.2, I3 de114 Second H 124R

AI de14, 12, 13 de114 Second H124L A2 de14, 13, 14 de112 Second V66L

G128 A1 de14, 5, 12, 13 (D83G)

Second H 124L

H124L G106 AI de15 de16 Second (N118D)

A1 12 G1 de16, I1 Same och137Q (L137och) A1 de16, I1 Same och137K

A2 de16 Same och137K A3 de16 Same och137L A4 de16 Same och137K

"The letter designates the a-thiophosphate nucleotide used to induce the second mutation that restores nuclease activity.

Typically, in those crosses in which nuc- recombinants were obtained, they represented approximately 10-25% of all of the transformants. Since nuc- plasmids result from the in vivo repair of heteroduplex molecules containing an intact strand from the NUC' isolate, all nuc- recombinant transformants initially must have been heterozygotes (see Figure 1C) that subsequently lost the genotype responsible for the NUC+ phenotype by either mismatch correc- tion or strand loss (PUKKILA et al. 1983). This conclusion was confirmed by forming ungapped heteroduplexes between ClaI-cleaved nucG153 plasmid DNA which was EcoK unmodified and PstI-cleaved wild-type pFOG301 (EcoK modified). Of 345 ampicillin-resistant transformants of strain 1 100 (EcoK restriction-plus) obtained with this mixture of heteroduplexes, 5 1 (or 15%) were tight nuclease-minus colonies. Control experiments with linear nucG153 DNA alone or linear wild-type DNA alone gave no transformants. From these results it is clear that the information present on one of the two strands of a heteroduplex plasmid is frequently lost immediately after DNA transformation in E . coli, allowing recessive markers generated by recombination to be readily expressed.

After localizing the second mutation which restores nuclease activity by a series of heteroduplex crosses to a defined physical interval, the nucleotide

550 D. SHORTLE AND R. LIN

sequence of the complete interval was determined. As shown in Table 2, true revertants were isolated for nucG131, nucAl21 and nucAll2 after misincorporation of an a-thiophosphate nucleotide which would be expected to revert the original mutation back to wild type. The most surprising result was the isolation of the same suppressor mutation sup124L for all four of the missense mutations. In addition, a second suppressor resulting in a different amino acid substitution at this same site, sup124R, was isolated from two of the four missense mutations (nucG131, nucA121), and a suppressor at another site, sup66L, was recovered for just one mutant (nucA121). Not surprisingly, all of the revertants of the ochre mutation nucA112 changed this stop codon to a translatable codon.

I t is worth noting that all 15 of these NUC+ revertant mutations have single base substitutions consistent with a single misincorporation of the a-thiophosphate nucleotide added to the first of the two gap misrepair reactions. Fur- thermore, the frequency of suppressor mutations falling within codon 137, which represents a target of 3 bp of a total of 4100, was in one case one per 5000 colonies and in the other 4 per 7000. True revertants, which would have a target size of one nucleotide out of 8200, were recovered at codons 76 and 79 at frequencies of one per 5000 and one per 10,000 colonies, respectively. These results indicate that the mutagenesis strategy used to both induce and revert nuc- mutations is quite efficient and yields a distribution that is close to random.

Suppression spectrum of supl 24L, supl 24R and sup66L: The codon substitutions of leucine or arginine for histidine at position 124 are able to suppress more than one nuc- mutation at different sites in the gene. Since the indirect mechanism responsible for the NUC- phenotype of the suppressible mutations is not known, no specific conclusions can be drawn at this point about the mechanism of this allele-nonspecific or “global” suppression. However, the fact that all four of the missense mutants reverted proved to be suppressible by at least one of the suppressors suggests that many of the “indirect” mutants may share a common defect. Before extending this type of reversion analysis to other nuc- alleles, it was clearly important to determine just how many of the nuc- mutations are suppressible by these three second-site suppressors. To use the deletion gap heteroduplex strategy to recombine the suppressor mutations with other nuc- alleles as outlined in Figure lD, a linker-bracketed deletion that removed the suppressed nuc- mutation but retained the suppressor had to be constructed from one of the isolated plasmids. This end, a segment of the nuc gene extending from the unique HpaI site at codon -34 to the unique HindIII site at codon 101, and thus containing the nucG131 mutation, was excised from nucG131 revertant plasmids G1 and A2 and a BamHI linker inserted in its place. The resulting deletion plasmids, de151 and de152, could then be used to cross the suppressor mutations sup124L and sup124R, respectively, into nuc- alleles between +1 and +loo. No convenient restriction sites were available for separating sup66L from the nucA121 mutation in isolate A2, so deletion de153 was generated by nuclease Ba131 digestion of HindIII linears of nucAl21 revertant plasmid A2, followed by insertion of a BamHI linker.


TABLE 3

Suppression spectrum of sup1 24L, sup124R and sup66L

55 1

nuc- allele 124L 124R 66L nuc- allele 124L 124R 66L

A105 A113 A121 A126 GI06 GI11 GI 16 G119 GI20 G121 G128 G131 C205 C207 C214 C216 c221

+ + +

ND + - + +

+ ‘r +

N D +

N D ?

-

- N D +

N D N D N D N D N D

N D N D + + +

N D N D N D N D N D +

N D N D N D N D N D N D

C222 C223 C226 C227 T202 T203 T204 T207 T211 T213 T215 T217 T222 T224 T228 T235

+ -

+ +

N D +

N D + + -

-

- +

N D N D N D N D N D N D N D N D N D N D N D N D N D N D N D N D

N D N D N D N D + +

N D +

N D N D N D N D N D N D N D

-

~~ ~ ~

Suppressor activity was scored by comparing the level of nuclease activity of each mutant when crossed with de151 (sup124L), de152 (sup124R) or de153 (sup66L) relative to a control cross with a deletion carrying the corresponding wild-type codon 124 or 66 (either de112, de113 or de115). Suppression is defined as the appearance of a second, clearly distinguishable population of transformants in the testcross with a distinctly higher level of nuclease activity than the single mutant alone. + indicates suppression, ? indicates probable, although weak, suppression, - indicates no suppression and N D means the cross was not done.

This small deletion permits efficient recombination only with nuc- mutations in the physical interval between codons 78 and 121.

The results of a number of heteroduplex crosses carried out to determine the suppression spectrum of the three suppressors are listed in Table 3. A total of 15 nuc- mutations are clearly suppressible by sup124L, four of which are the same mutations reverted directly to give this codon change. Particularly noteworthy is the fact that all 14 mutant alleles in the “indirect” class tested which had activity levels of ++, + or +/- were suppressed by sup124L, whereas only two of 15 in this class with 0 activity levels were suppressed. In addition, the two mutant alleles in the catalytic and binding classes tested were not suppressed.

Fewer crosses were done with the relatively weak suppressor sup124R, but at least two additional suppressible alleles were identified. In addition, six more nuc- alleles suppressible by sup66L were also found. Unfortunately, the relatively small overlap between de151 and de153 meant that only some of the alleles suppressible by sup124L could be examined for suppression by sup66L. Nevertheless, in all four cases tested, each allele suppressible by sup124L was also suppressed by sup66L. By crossing de151 and de152 against de16 and by crossing de153 against wild-type plasmid pFOG30 1 (and sequencing several isolates), it was possible to recombine each of the three suppressor mutations into an otherwise wild-type gene. Each suppressor in isolation was indistinguish-

5 5 2 D. SHORTLE AND B. LIN

FIGURE S.-Positions of the a-carbons of the amino acid residues altered by the three global suppressor mutations and by the suppressible nut- mutations. This ribbon diagram of the three- dimensional structure of staphylococcal nuclease was drawn and copyrighted by JANE RICHARDSON (see RICHARDSON 1981) and is used with her permission.

able from wild-type plasmid when scored by the TBD agar replica plating assay. Since mock experiments with dilutions of purified nuclease indicate that this assay can reliably detect a three- to four-fold increase in total enzyme activity, it seems unlikely that the suppressors act via a large overproduction of protein.

Examination of the types of amino acid substitutions caused by the suppressible nuc- mutations does not reveal any obvious patterns. For instance, two mutations (nucA 113, nucT235) result in the substitution of the large hydrophobic residue phenylalanine for the smaller hydrophobic residue valine, whereas another mutation (nucG131) results in substitution of valine for phenylalanine. With nuc- allele C222, a surface lysine is replaced with uncharged asparagine, whereas a surface asparagine is replaced with the charged residue aspartic acid in nucGl06, and a relatively buried glutamic acid goes to lysine in nucT213.

In Figure 3 the relative spatial positions of the amino acid residues altered by the suppressible nuc- alleles and the suppressor mutations are shown sche- matically on a ribbon cartoon drawn by JANE RICHARDSON on the basis of the X-ray diffraction map of staphylococcal nuclease. Again, there is no obvious

GENETIC ANALYSIS OF STAPH NUCLEASE 553

pattern to the distribution of sites around the molecule. (The absence of mutant sites in the long a-helix from 121 to 141 is due to the inability to test mutations in this region for lack of the appropriate linker-bracketed deletions.) The positions of the amino acid changes produced by the suppressors sup124 and sup66 are essentially on opposite sides of the molecule. Although not apparent in this diagram, from three-dimensional models the wild-type histidine side chain at position 124 can be seen to lie flat against the surface of the protein, whereas at position 66, the wild-type valine side chain is completely buried within a core of hydrophobic residues.

CONCLUSIONS

The finding that the majority of tested mutations which alter amino acids remote from the active site are suppressible by one or more of the three suppressors suggests there may be a single mechanism behind the NUC- phenotype of this class of alleles. Perhaps the simplest explanation which would allow for the multiplicity of types and positions of amino acid changes produced by the suppressible nuc- mutations is that these substitutions exert their effect by destabilizing the native conformation and that the reduction in enzyme activity of each variant nuclease parallels this loss in stability.

In support of this idea are the correlations between residual nuclease activity and types of amino acid substitution discernable at several mutant sites involving hydrophobic residues. At codon 76, for instance, replacement of phenylalanine with tyrosine gives a moderate level of activity, whereas the less con- servative replacement with valine results in very weak activity, and replacement with serine eliminates all detectable activity. Similarly, for the leucine codons at 37 and 103, as the substituted amino acid becomes larger or less hydrophobic, the effect on enzyme activity increases. If the suppressible nuc- alleles do in fact encode less stable nuclease molecules, then the three global suppressors could increase the amount of scoreable nuclease by one of several classes of mechanisms. Those mechanisms that lead to an overproduction of protein seem unlikely given the apparently normal enzyme activity of the suppressors in a wild-type background and the lack of suppression of a leaky mutation which modifies an active site residue (nucG111).

T o determine whether the “indirect” class of mutations do code for proteins with folding/stability defects and whether phenotypic suppression by the three suppressor mutations results from the structural consequences of the amino acid changes at positions 66 and 124, experiments are now in progress to characterize purified mutant proteins containing amino acid substitutions spec- ified by nuclease genes with (1) nuc- mutations alone, ( 2 ) sup mutations alone and (3) various combinations of nuc- and sup mutations. If these biochemical experiments should demonstrate that the suppressors act by increasing the protein conformational stability, the suppression spectrum data presented in this report would rather clearly indicate that alterations in stability induced by a perturbation at one site on a protein molecule can, in some cases, be com- pensated by amino acid residue substitutions at remote sites on the same molecule.


T h e authors would like to thank GREG JAROSIK and MARSHA MCCONNELL for their contributions to the DNA sequencing of sonie of the mutant plasmids, STEVE HARRISON and GARY ACKERS for helpful discussions and STEPHEN BENKOVIC and FRITZ ECKSTEIN for their generous gifts of a- thiophosphate nucleotides. Financial support for this research was from National Institutes of Health grant GM 30649 and from an award by the Sinsheinier Foundation.

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Communicating editor: D. BOTSTEIN

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