THE JOURNAL OF BIOLOGICAL No. 25, pp. Printed in U.S.A ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 3, Issue of January 25, pp. 2075-2082,1993 Printed in U.S.A. Expression

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 3, Issue of January 25, pp. 2075-2082,1993 Printed in U.S.A.

Expression of Drosophila Rrpl Protein in Escherichia coli ENZYMATIC AND PHYSICAL CHARACTERIZATION OF THE INTACT PROTEIN AND A CARBOXYL- TERMINALLY DELETED EXONUCLEASE-DEFICIENT MUTANT*

(Received for publication, April 10, 1992)

Miriam Sander$, Meryl Carter, and Shu-Mei Huang From the Laboratov of Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Drosophila Rrpl protein purified from embryos has four tightly associated enzymatic activities: DNA strand transfer, single-strand DNA renaturation, 3’- exonuclease, and apurinic endonuclease. Copurifying with these activities is a single polypeptide that has an apparent M, of 105,000 when estimated by SDS-polyacrylamide gel electrophoresis. To determine if this polypeptide is sufficient for these activities, it has been overexpressed in Escherichia coli. In crude extracts of E. coli cells, an ATP-independent Mg2+-dependent strand transfer activity is observed upon activation of the promoter that drives expression of Rrpl. Rrpl protein purified from induced E. coli cells has electrophoretic, chromatographic, and enzymatic properties similar to those of Drosophila Rrpl protein.

The carboxyl-terminal region of Rrpl (amino acids 428-679) is homologous to E. coli exonuclease 111. Rrpl deleted for this region cannot carry out DNA strand transfer, but can renature complementary single-strand DNA. The strand transfer activity of this truncated protein can be restored if DNA 3’-exonuclease is provided in trans by pretreating the double- strand DNA substrate with E. coli exonuclease 111. This demonstrates a likely role of the exonuclease in the in vitro DNA strand transfer reaction carried out by Rrpl protein. Such a role is also suggested by an analysis of the polarity of the strand transfer reaction.

The enzymology of DNA repair and homologous recombination has been studied in great detail in prokaryotes (for reviews, see Refs. 1-3). Our knowledge of the enzymes involved in similar pathways in eukaryotic species is not as well developed. However, rapid progress has been made recently in studies of the yeast Saccharomyces cereuisiae (4, 5 ) . By analogy to the prokaryotic systems, it was expected that homologous recombination in eukaryotes would require a protein that promotes joint molecule formation and DNA strand transfer between homologous DNA molecules, that is, a protein analogue of Escherichia coli recA protein. Candidates for such a protein have been characterized in yeast, Drosoph- ila, and human cells (Refs. 6-11; for review, see Ref. 12). These proteins, identified by in vitro activity assays, differ from recA protein in many respects. For example, the yeast and Drosophila protein sequences do not show homology to prokaryotic recombination proteins (13, 14). However, inde-

* 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 U.S.C. Section 1734 solely to indicate this fact.

$ T o whom correspondence should be addressed Laboratory of Genetics, NIEHS, P. 0. Box 12233, Mail Drop D3-04, Research Triangle Park, NC 27709. Tel.: 919-541-2799; Fax: 919-541-7593.

pendent work recently identified two proteins from yeast whose amino acid sequences are homologous to the recA protein sequence (15, 16). The interactions between these yeast proteins, which have potentially overlapping functions in uiuo, are not yet well characterized.

A number of in vitro assays for homologous recombination have been developed. One of the most extensively used systems utilizes homologous linear dsDNA’ and circular ssDNA molecules (17-19). Using these model substrates, studies with recA protein define three phases in the strand transfer process (20). In the first phase of the reaction, presynapsis, recA protein forms a stoichiometric complex with ssDNA molecules. In the second phase, synapsis, homologous associations are made between ssDNA and dsDNA molecules, and three- strand structures known as joint molecules are formed. Joint molecules are extended by branch migration and strand displacement during the third and final phase of the process. During this last phase, extensive heteroduplex regions can be formed as a consequence of the strand displacement process. However, it has not been established yet that this paradigm for strand transfer reactions is applicable to the reactions carried out by eukaryotic strand transfer proteins.

Using a sensitive in vitro assay for DNA strand transfer that detects the formation of both joint molecules and strand transfer products, one major strand transfer activity is found in extracts of Drosophila embryos (9,21). In a highly purified fraction, a single polypeptide copurifies with the strand transfer activity (9). Enzymatic characterization of this fraction demonstrates that ssDNA renaturation, 3’-exonuclease, and apurinic (AP) endonuclease activities are tightly associated with the single protein species (22). This protein has been named Rrpl (recombination repair protein 1). Whereas its biochemical characteristics in vitro suggest possible roles in both homologous recombination and DNA repair, the biological functions of Rrpl in uiuo are not yet known.

The gene for Rrpl has been characterized (14). Analysis of the DNA sequence revealed a probable bipartite organization of the protein it encodes. The NHp-terminal portion is lysine- and glutamate-rich and is not related to other known proteins. The COOH-terminal portion is homologous to the DNA repair endo/exonuclease E. coli exonuclease 111. This homology im- plies that the COOH-terminal region of Rrpl is likely to be responsible for the nuclease activities that copurify with the strand transfer activity.

In this work, the enzymatic, chromatographic, and electrophoretic properties of recombinant Rrpl protein expressed in E. coli are examined. These experiments demonstrate that the single polypeptide encoded by the Rrpl gene is sufficient for

The abbreviations used are: dsDNA, double-strand DNA; ssDNA, single-strand DNA; AP, apurinic/apyrimidinic; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kb, kilobase(s); IPTG, isopropyl-1-thio-(3-D-galactopyranoside.

2075

2076 Recombinant Rrpl Protein Is Multifunctional

four enzymatic activities: DNA strand transfer, ssDNA renaturation, 3’-exonuclease, and AP endonuclease. The properties of a COOH-terminally deleted form of Rrpl protein suggest that the exonucleolytic processing of dsDNA substrates is involved in the initiation of the in vitro strand transfer reaction.

EXPERIMENTAL PROCEDURES

Nucleic Acids and Enzymes-Plasmid DNA substrates were purified by an alkaline lysis procedure (23) followed by chromatography on Qiagen (Qiagen, Inc). Bacteriophage ssDNA was purified by Qia- gen chromatography. Labeled dsDNA substrates were prepared using standard methods (24). Reagents and enzymes were obtained from the following sources: pETSa, pETJd, and E. coli host strains, Nov- agen; T4 DNA polymerase, T4 polynucleotide kinase, and exonuclease 111, New England BioLabs, Inc.; restriction enzymes, New England BioLabs, Inc. or Life Technologies, Inc; AmpliTAQ DNA polymerase, Perkin-Elmer Cetus Instruments; bacterial alkaline phosphatase, Life Technologies, Inc.; and calf thymus histone H1 and E. coli recA protein, Sigma. Rrpl protein was purified from Drosophila embryos as described (9). E. coli single-strand binding protein was a generous gift of M. M. Cox (University of Wisconsin).

Preparation of Resected DNA Substrate-Resected DNA was prepared by treating the 540-bp DdeI fragment of pBluescript SK(-) 5’- “‘P-end-labeled on both strands with exonuclease 111. The product was purified by phenol extraction followed by ethanol precipitation. The average length of the 5’-overhang was determined by denaturing PAGE in the presence of size standards.

Strand Transfer Assay-Strand transfer activity was assayed by the method of McCarthy et al. (21). Strand transfer reactions were carried out in 10-pl volumes containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCI2, 50 pg/ml bovine serum albumin, 12 ng of pBluescript SK(-) ssDNA, and -0.25 ng of a homologous dsDNA fragment 5’- “‘P-end-labeled on both strands. Either the 540- or 409-bp DdeI restriction fragment of pBluescript SK(-) was routinely used as the dsDNA substrate. dsDNA fragments were always less than equimolar with the circular ssDNA substrate. Reactions were initiated by addition of enzyme; incubated for 10 min at 37 “C; and terminated by the addition of SDS, EDTA, and proteinase K to concentrations of 0.5%, 1 2 mM, and 15 pg/ml, respectively. After incubation at 42 “C for 10 min, samples were analyzed by agarose gel electrophoresis in Tris/ borate/EDTA buffer. One unit of strand transfer activity is the amount of enzyme required to convert >50% of the labeled substrate to product under the conditions described here. Because of the ex- treme cooperativity of both strand transfer and ssDNA renaturation reactions, the critical variable used in determining activity levels is protein concentration (9).

This assay tests for an activity that can pair duplex DNA with homologous single-strand DNA under conditions in which this pairing does not occur spontaneously. However, the term DNA strand transfer as used here does not imply a specific product structure. In this context, “strand transfer product” refers to both joint molecules (three-strand DNA species that have not undergone a complete strand displacement) and two-strand DNA species from which one strand of the parental duplex has been released by strand displacement.

ssDNA Renaturation Assay-ssDNA renaturation activity was measured under strand transfer assay conditions, except that incubation was for 10 min at 30 “C. The ssDNA substrates for renaturation were the homologous strands of the 166-bp DdeI fragment of pBluescript SK(-) .!~’-~‘P-end-labeled on both strands. The 166-bp fragment was purified by preparative acrylamide gel electrophoresis and subsequently denatured by boiling immediately prior to use. Samples were analyzed by agarose gel electrophoresis, and renaturation was monitored by following the electrophoretic mobility shift of both single strands that results from formation of duplex DNA. Background spontaneous renaturation was 52%. When required, quantitative densitometry was performed on the autoradiographs of dried gels.

3’-Exonuclease Assay”3’-Exonuclease activity was assayed using two methods. The first method is semiquantitative and was used to analyze column fractions. This assay measures the loss of 3’-end- specific 32P label incorporated at the terminus of a blunt-end 7-kb dsDNA fragment. This substrate was prepared by T4 DNA polymerase treatment of SmaI-digested pMS215 (14) in the presence of [a- :”P]dCTP (24). As an internal control, the 540-bp 5’-32P-labeled DdeI fragment of pBluescript SK(-) was also included in each assay. The reaction was carried out in a 1O-pl volume containing 20 mM Tris-

HCI, pH 7.5,lO mM MgC12, 50 pg/ml bovine serum albumin, and -1.0 and ~ 0 . 2 ng of the 3‘- and 5’-*’P-labeled fragments, respectively. Samples were incubated for 10 min at 37 “C. The specificity of the exonuclease was determined by comparing the relative loss of 32P label from the two fragments in the same assay. Reaction products were resolved by agarose gel electrophoresis, and samples were quan- titated by densitometric analysis of autoradiographs.

A second more quantitative 3’-exonuclease assay was used to determine the specific activity of protein fractions. An 800-bp dsDNA fragment was uniformly labeled with [3H]thymidine to a specific activity of 6.4 X lo6 cpmlpg by a polymerase chain reaction method that will be described elsewhere? (The polymerase chain reaction primers used to generate this substrate are identical to those used to construct plasmid pRrpl-C259, as described below.) The exonuclease reaction was carried out in a 20-pl volume containing 20 mM Tris- HC1, pH 7.5, 10 mM MgC12, 50 pg/ml bovine serum albumin, and 8 ng of the substrate DNA. Reactions were incubated at 30 “C. At appropriate time points, the reaction was terminated by the addition of 80 pl of 20% trichloroacetic acid containing 50 pg/ml salmon sperm DNA. After incubation for 30 min on ice, the samples were centrifuged in a microcentrifuge for 15 min at 4 “C. 75 pl of the supernatant fraction was removed and added to Ecolite scintillation fluid (ICN Biochemicals). The amount of trichloroacetic acid-soluble radioactiv- ity was determined using a Beckman LS 7800 liquid scintillation counter. One unit of 3’-exonuclease activity releases 1 nmol of trichloroacetic acid-soluble nucleotides in 30 min at 30 “C.

AP Endonuclease Assay-AP endonuclease activity was analyzed as described previously (22). Partially depurinated supercoiled pBluescript SK(-) plasmid DNA (100-150 fmol) was preincubated in 20 pl of buffer containing 50 mM Tris-HCI, pH 7.5, 5 mM MgClz, and 50 pg/ml bovine serum albumin for 2 min at 30 “C. Reactions were initiated by the addition of enzyme and incubated at 30 ‘C. Electrophoresis of reaction products was carried out in Trislacetatel EDTA buffer. Photographic negatives of the agarose gels were quan- titated by densitometry. Quantitation was carried out on reactions performed in duplicate. One unit of activity is defined as the amount of protein required to produce a rate of AP site cleavage of 1 pmol/ min.

Purification of Rrpl Protein-A typical preparation started with a 2-liter culture of induced E. coli cells. LB medium was inoculated with 0.008 volume of a fresh overnight culture and grown at 37 “C to mid-log phase. IPTG was added to 0.4 mM and incubated at 37 “C for 3 h. Cells were collected by centrifugation at 4000 rpm for 45 min at 4 “C in a Sorvall HG-4L rotor. All subsequent steps were carried out at 4 “C. Cells were washed with 0.1 volume (200 ml) of cold Tris/ EDTA buffer and recentrifuged at 4000 X g for 10 min. The cell pellet was resuspended in 0.5 volume (100 ml) of buffer S1 (10% glycerol, 50 mM Hepes, pH 7.5, 1.0 mM EDTA, 0.4 mM dithiothreitol, 0.1% Triton X-100, 1.0 mM phenylmethylsulfonyl fluoride, and 0.50 pg/ml each of the peptides pepstatin A, leupeptin, chymostatin, antipain, and aprotinin) containing 0.1 M NaCl by several passes with the A- pestle of a Dounce homogenizer. Cells were frozen in a dry ice/ethanol mixture for at least 10 min and thawed in an ice-water bath, and the insoluble material was collected by centrifugation (23,000 X g, 20 min). The supernatant was removed, and the pellet was resuspended in an equal volume (100 ml) of buffer S1 containing 0.6 M NaCl by pipetting. The sample was centrifuged in a Sorvall Ti-60 rotor for 40 min at 48,000 rpm. The supernatant was removed (Fraction I) and diluted with an equal volume of buffer S2 (10% glycerol, 50 mM Hepes, pH 7.5,O.l mM EDTA, 0.2 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 0.25 pg/ml each of the peptides pepstatin A, leupeptin, chymostatin, antipain, and aprotinin) containing 0.1% Triton X-100. This sample was applied to a 5-ml Bio-Rex 70 column equilibrated with buffer S2 containing 0.3 M NaCl at a flow rate of 0.8 ml/min. The column was washed with 20 ml of buffer S2 containing 0.3 M NaCl and eluted with a 100-ml gradient from 0.3 to 1.0 M NaCI. Rrpl elutes in a single peak at 0.45 M NaCI. The active fractions were pooled (Fraction 11) and concentrated by a Centricon-30 device (Fraction IIc). Fraction IIc, containing 0.6 mg of protein in a volume of 0.7 ml, was adjusted to 0.05 M NaCl with 9 volumes of buffer S2 and applied to a 2-ml heparin-Sepharose column equilibrated with buffer S2 containing 0.05 M NaCl at a flow rate of 0.4 ml/min. The column was washed with 10 ml of buffer S2 containing 100 mM NaCl and eluted with a 100-ml gradient from 0.1 to 2.0 M NaCI.

lose membranes (BA83, Schleicher & Schuell) using the Milliblot- Immunoblotting-Protein samples were transferred to nitrocellu-

M. Sander, M. Carter, and S-M. Huang, manuscript in preparation.

Recombinant Rrpl Protein Is Multifunctional 2077

SDE transfer system as specified by the manufacturer. The blot was then processed using a Vectastain ABC-alkaline phosphatase kit. Rabbit antiserum to a peptide corresponding to Rrpl amino acids 499-517 was a generous gift of Dr. Ky Lowenhaupt. The antiserum was diluted 5000-fold in the experiment shown in Fig. 2.

Analysis of Proteins by SDS-PAGE-Proteins were analyzed using standard conditions of SDS-PAGE (25). The marker proteins used were myosin, @-galactosidase, phosphorylase b, bovine serum albumin, and ovalbumin (9% gels) or phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lyso- zyme (12% gels).

Plasmid Construction-Plasmids expressing truncated forms of Rrpl were constructed as follows. pRrpl-El was digested to comple- tion with EcoRI, and the 5.0- and 0.35-kb fragments were isolated by preparative agarose gel electrophoresis followed by electroelution. The 5.0-kb fragment was self-ligated to make pRrpl-N452. The 5.0- and 0.35-kb fragments were ligated together and screened for fragment orientation to make pRrpl-N551. The inserts of pRrpl-C289 and pRrpl-C259 were generated by polymerase chain reaction. The 5'-oligonucleotide primers were designed to insert an NcoI site immediately adjacent to coordinates 1307 and 1397 for plasmids pRrpl- C289 and pRrpl-C259, respectively. (Coordinates refer to the Rrpl cDNA sequence (14).) The 3'-oligonucleotide primer for both con- tructs inserted a BamHI site immediately adjacent to coordinate 2191. The polymerase chain reaction fragments were digested with NcoI and BamHI and cloned into vector pETBd, also restricted with NcoI and BamHI.

The analysis of protein expression from plasmid constructs was carried out as described previously (14).

Other Methods-Protein molecular weights were predicted from deduced amino acid sequences using the Peptidesort program of the University of Wisconsin Genetics Computer Group sequence analysis software (26). Protein concentrations were determined by the Coo- massie dye binding method of Bradford (27). Densitometric analysis was carried out using a Pharmacia LKB Biotechnology Ultrascan XL densitometer.

RESULTS

Expression of Rrpl Protein in E. coli: Electrophoretic Prop- erties and Antibody Reactivity-To express the Rrpl polypeptide in E. coli, the Rrpl cDNA was subcloned into the pET3a vector (14). This vector provides for expression utilizing a T7 RNA polymerase promoter. An IPTG-inducible source of T7 RNA polymerase is provided on the bacterial chromosome of the host strain (28). The plasmid constructs used in this work are shown schematically in Fig. 1. The plasmid pRrpl-El fuses the entire Rrpl open reading frame to a 14-amino acid leader peptide synthesized from the T7 RNA polymerase promoter (14). A plasmid with the Rrpl cDNA in reverse orientation was isolated for use as a control in some experiments. Four additional plasmids were constructed that produce deleted forms of Rrpl protein. Two of these plasmids, pRrpl-N551 and pRrpl-N452, produce proteins that are COOH-terminally deleted; they include the first 551 or 452 amino acids of Rrpl protein, respectively. Two others are NH2-terminally deleted; pRrpl-C289 and pRrpl-C259 produce proteins that include the last 289 or 259 amino acids of Rrpl, respectively. Each of the proteins synthesized from these plasmids will be referred to by its length in number of amino acids (see Fig. 1). Native Rrpl protein is called Rrpl- 679, and the full-length recombinant protein is Rrpl-693.

Fig. 2 shows protein gel and immunoblot analyses of the proteins present in E. coli cells carrying four of these plasmids. The first lune shows the proteins present in a culture carrying the plasmid pRrpl-El prior to induction of the T7 RNA polymerase promoter. The remaining samples were prepared after incubation of the cultures in the presence of IPTG. Comparison of the first lune with the second through fifth lanes shows the IPTG-dependent appearance of a single major protein species in each culture. An immunoblot analysis of the same samples was carried out using an antipeptide antibody specific for Rrpl amino acids 499-517. A strong and specific reactivity with the induced bands in the second, third,

A.

pRfpl-El 7933 bp Rrp1-693 693 a8

- PET vector

D TI promoter

0 Rrpl cDNA

Nuclease domain

1 14 aa leader pepllde

- x

AT0 TAA

pRrpl-E9 TAA ATG

mxa amp ori

B.

FIG. 1. Diagrams of plasmids used in this study. A, two plasmids containing the entire Rrpl cDNA and an adjacent portion of Xgtll are shown. The forward construct, pRrpl-El, synthesizes the full-length 693-amino acid (a) recombinant Rrpl protein, Rrpl-693. The reverse construct, pRrpl-E9, synthesizes a protein encoded by the XDNA, Rrpl-Rev. The positions of the start and stop codons of the Rrpl open reading frame are indicated. B, plasmids used to synthesize truncated Rrpl proteins are shown. The name of the protein produced from each plasmid is indicated directly above each plasmid and indicates the length of the protein in amino acids. The origin region (ori) and ampicillin resistance gene (amp) are present, but not indicated in B. Maps in both A and B are not drawn to scale.

-- origln _, .

116 - 97 -

66-

45 - 29-

- 116 - 97

FIG. 2. Protein gel and immunoblot of recombinant Rrpl proteins. Cultures carrying the plasmids expressing the indicated proteins were grown to mid-log phase. An aliquot was taken for the preinduction control, and the remaining culture was then induced with 0.4 mM IPTG for 3 h. 1-ml aliquots of the induced cells were centrifuged in a microcentrifuge, and the cell pellets were resuspended in 50 pl of H20 followed by addition of 50 pl of a solution containing 10% glycerol, 75 mM Tris-HC1, pH 6.8, 2% sodium dodecyl sulfate, 100 mM dithiothreitol, and 0.1% bromphenol blue. 20 pl of each sample was analyzed by SDS-PAGE (25) on a 9% polyacrylamide gel and visualized by Coomassie Blue staining (left). Identical samples were transferred to nitrocellulose and visualized by immunoblotting with antipeptide antisera specific to Rrpl amino acids 499-517 (right). The samples loaded are as follows (left to right): uninduced cells and induced cells expressing Rrpl-693, Rrpl-N551, Rrpl-N452, or Rrpl-C259. The mobilities of marker proteins (in kilodaltons) and the origin of the resolving gel are indicated.

2078 Recombinant Rrpl Protein Is Multifunctional

and fifth lanes is observed, indicating that these proteins are in fact Rrpl-related proteins. The induced protein in the fourth lane does not react with this antibody, as expected, since this deleted protein includes only the first 452 amino acids of Rrpl and therefore lacks the peptide epitope.

When calculated from its amino acid sequence, the predicted molecular weight of intact native Rrpl protein (Rrpl- 679) is 74,500. However, when estimated by electrophoretic mobility during SDS-PAGE, the apparent M, of the polypeptide is 105,000. An aberrant electrophoretic mobility is also observed for recombinant Rrpl protein (Rrpl-693) expressed in E. coli (Table I). The predicted M , of Rrpl-693 is 76,043, but the observed M , during electrophoresis is 102,000. The ratio of the predicted M, to the observed M , estimates the degree of retardation during electrophoresis. Table I shows that the COOH-terminally deleted Rrpl protein species, Rrpl-N551 and Rrpl-N452, are retarded during electrophoresis as much as the intact protein (ratios of 0.74, 0.70, and 0.74, respectively). However, deletion of part (Rrpl-C289) or all (Rrpl-C259) of the NH2-terminal region partly (0.77) or nearly completely (0.91) restores normal electrophoretic mobility. This result suggests that the NH2-terminal lysine/ glutamate-rich region of Rrpl protein is responsible for its aberrant behavior during electrophoresis, as suggested previously (14).

The enzymatic properties of the proteins Rrpl-693, Rrpl- N551, and Rrpl-N452 are characterized in the following experiments. The NH2-terminally deleted proteins Rrpl-C289 and Rrpl-C259 were not studied further since it was not possible to obtain them in an active soluble fraction.

Enzymatic Properties of Recombinant Rrpl Protein-Par- tial purification of Rrpl-693 was achieved by gentle lysis of induced E. coli cells and successive extractions in the presence of buffers containing 0.1, 0.6, and 1.0 M NaCl. The protein extracts obtained from a culture expressing Rrpl-693 are shown in Fig. 3 (lanes 1-3 and 7). The induced polypeptide is somewhat enriched in the 0.1 M fraction (lane 1) and greatly enriched in the 0.6 M fraction (lane 2). Control extracts lacking Rrpl-693 are shown in lanes 4-6 and 8. (The control culture used in this experiment expresses protein from the reverse plasmid construct pRrpl-E9 shown in Fig. 1.) Each of the fractions shown in lanes 1-6 was tested in a strand transfer assay (Fig. 4). The results show a strong correlation between the abundance of Rrpl-693 protein and the amount of strand transfer activity. The control fractions that lack Rrpl show no detectable activity (Fig. 4B), whereas the fraction highly enriched for Rrpl-693 is most active (Fig. 4A). The activity is not stimulated by ATP and is MP-dependent (data not shown).

This in vitro strand transfer reaction assays for the ability

TABLE I Electrophoretic mobility of intact and deleted forms of Rrpl

Protein predicted" M. obsewedb M, Predicted/obsewed

Rvl-679 74,500 105,000 0.71 Rvl-693 76,043 102,000 0.74 Rrpl-N551 59,450 80,000 0.74 Rrpl-N452 48,062 68,000 0.70 Rrpl-C289 33,226 43,000 0.77

The molecular weight of each protein species was calculated using the amino acid sequence deduced from the cDNA (Rrpl-679) or plasmid construct (all others) (see Fig. 1).

*The indicated molecular weight is the value observed during denaturing gel electrophoresis. The electrophoretic mobility of each protein was compared to the mobilities of globular marker proteins during SDS-PAGE using either 9% (Rrpl-679, Rrpl-693, Rrpl-N551, and Rrpl-N452) or 12% (Rrpl-C289 and Rrpl-C259) resolving gels.

Rrpl-C259 30,077 33,000 0.91

$ ,@ ,1c' \1c' 4 9

Q e" Q e" e-" plasmld e& Q Q

0.1 0.6 1.0 0.1 0.6 1.0 1.0 1.0 (W

"2(H

- 116

- 97

- 66

- 45

1 2 3 4 5 6 7 8

FIG. 3. Partial purification of Rrpl-693. Mid-log phase cultures carrying either pRrp1-El (lanes 1-3 and 7) or pRrpl-E9 (lanes 4-6 and 8 ) were induced with 0.4 mM IPTG for 3 h. The induced cells were collected by centrifugation, washed, and then lysed by a single freeze/thaw cycle in buffer SI containing 0.1 M NaC1. The cell debris was removed by centrifugation, and the pellet fraction was re- extracted sequentially with buffer S1 containing 0.6 and 1.0 M NaC1. The remaining pellet was extracted with SDS-polyacrylamide gel loading buffer. Aliquots of the supernatant protein fractions (lanes 1-6) and pellet protein fractions (lanes 7 and 8) were analyzed by SDS-PAGE on an 8% polyacrylamide gel. The mobilities of markers and their sizes (in kilodaltons) are indicated.

A* 1 2 3 0.1 M O.6M 1.011 NaCl

I rn II i ."- *".lpTp-

-ST product

I -subatrate

B.

0 eana-w -aubshate uu-

4 5 6 - 0.1 M 0.6M 1.OM - NaCl

FIG. 4. Rrpl-693 strand transfer activity in partially purified fractions. Strand transfer ( S T ) assays were carried out under standard conditions. The numbers 1-6 indicate addition of the samples analyzed in lanes 1-6 of Fig. 3. For each sample assayed, a series of reactions were performed with serial 2-fold dilutions of the enzyme. Each series is shown with enzyme amount decreasing from left to right. A , enzyme samples were from a culture carrying the plasmid pRrpl-El expressing protein Rrpl-693. The highest amounts of protein added were as follows: 1-3, 0.90, 0.45, and 0.16 pg, respectively. B, enzyme samples were from a culture carrying the plasmid pRrpl- E9 expressing protein Rrpl-Rev. The highest amounts of protein added were as follows: 4-6, 1.50, 0.52, and 0.36 pg, respectively.

of a protein to pair duplex DNA with homologous single- strand DNA under conditions in which this pairing does not occur spontaneously. Results obtained using this assay confirm the properties of the reaction established previously using other assays and other strand transfer proteins (21). However, the term DNA strand transfer as used here does not imply a specific product structure; the assay does not distinguish between products that are joint molecules (three-strand DNA

Recombinant Rrpl Protein Is Multifunctional 2079

species that have not undergone a complete strand displacement) and those that are two-strand DNA species from which one strand of the parental duplex has been released by strand displacement.

Purification of Recombinant Rrpl-The assay results described above suggest that Rrpl-693 protein is sufficient for the strand transfer reaction. To confirm this suggestion, the partially purified 0.6 M NaCl fraction was further purified using two ion-exchange chromatography steps, Bio-Rex 70 and heparin-Sepharose. Column fractions from the heparin- Sepharose chromatography step were analyzed for strand transfer activity. A single activity peak is observed that cor- responds closely with the elution profile for Rrpl protein (Fig. 5). Assays for ssDNA renaturation and 3’-exonuclease activities give very similar results: single activity peaks are observed that coelute with Rrpl protein. Similar results were obtained when Rrpl-679 purified from Drosophila embryos was chro- matographed on either ssDNA-agarose or Monos resin (22).

The specific activities of a number of Rrpl-693 protein fractions in the strand transfer reaction were compared (Table 11). Fraction I (0.6 M NaCl extract) is -5-fold lower in specific activity than Fraction I1 (Bio-Rex 70). This specific activity change reflects the removal of protein contaminants by the Bio-Rex 70 column step (data not shown). Additional purification by chromatography on heparin-Sepharose (Fraction 111-HS) or Sephacryl S-400 (Fraction 111-S-400) did not sig-

A

e4

?D

0 0

fnction no

+ ” ” ” f + + + + - - -

origin - . .

205 - 116-

97 - 66-

45 -

lractlon

acllvlty

e Rrp1-693

FIG. 5. Heparin-Sepharose chromatography of Rrpl-693. Rrpl-693 was purified through the Bio-Rex 70 chromatography step (Fraction 11) and applied to a 2.0-ml heparin-Sepharose column (see “Experimental Procedures”). Fractions were assayed for strand transfer, ssDNA renaturation, and 3’-exonuclease as described under “Experimental Procedures.” Analysis of the first 40 ml of the gradient are shown. A, elution patterns for strand transfer (O), ssDNA renaturation (O), and 3’-exonuclease (0) activities are shown; R, aliquots of the indicated fractions were analyzed on a 9% gel by SDS-PAGE. The mobilities of marker proteins (in kilodaltons) and the origin of the resolving gel are indicated.

nificantly increase the specific activity of the fraction or its purity as judged by gel electrophoresis (Table 11) (data not shown). Fractions I1 and I11 of Rrpl-693 were also tested in a quantitative 3’-exonuclease assay, and their specific activities were compared. Similarly, the three fractions demonstrate similar specific activities in this assay. AP endonuclease activity was previously reported to be associated with Rrpl-679 (22). A highly active AP endonuclease was also associated with recombinant Rrpl-693 (Table 11).

Furthermore, the strand transfer-specific and AP endonuclease-specific activities of recombinant Rrpl-693 are very close to the values for Rrpl-679 purified from Drosophila (Table 11). This suggests that a native-like Rrpl protein is synthesized in E. coli that is not deficient in any cofactor or covalent modifications required for enzyme function. How- ever, the 3’-exonuclease-specific activity of Rrpl-693 is -4- fold higher than that measured for Rrpl-679. The significance of this result is not known.

Partial Purification of COOH-terminally Truncated Rrpl- N551 Protein-Using partially purified fractions, we tested the ability of the two COOH-terminally deleted Rrpl proteins, Rrpl-N551 and Rrpl-N452, to carry out DNA strand transfer reactions. Both proteins were deficient in strand transfer, but were able to carry out ssDNA renaturation (data not shown). Rrpl-N551 protein was then further purified by Bio-Rex 70 chromatography; and the column fractions were assayed for DNA strand transfer, ssDNA renaturation, and 3’-exonuclease. The results are shown in Fig. 6. A single peak of Rrpl- N551 protein elutes from the column a t -0.55 M NaCl (Fig. 6B). Coeluting with this protein is a peak of ssDNA renaturation activity (Fig. 6A). No peak of strand transfer or exonuclease activity is associated with the peak of Rrpl-N551 protein. Thus, specific alteration of the expressed protein by truncation has altered its enzymatic properties, providing further confirmation that the exonuclease and strand transfer activities depend on the function of intact Rrpl protein. Quantitative exonuclease assays were used to estimate the 3’- exonuclease-specific activity of Rrpl-N551 protein after Bio- Rex 70 chromatography (Table 11, Rrpl-N551, Fraction 11). An extremely low level of activity was observed, indicating a specific activity -100-fold lower than that of Rrpl-693.

Exonuclease-deficient Rrpl-N551 Pairs ssDNA with Par- tially Single-strand dsDNA-The properties of Rrpl-N551 show that deletion of the COOH-terminal 142 amino acids of Rrpl is correlated with the loss of both exonuclease and strand transfer activities. To test the role of the exonuclease in the strand transfer reaction, we examined the ability of Rrpl- N551 to form joint molecules between partially single-strand linear dsDNA and homologous circular ssDNA. The dsDNA substrate was predigested to a limited extent with E. coli exonuclease 111. The products of this reaction were 3”resected DNA molecules with ssDNA 5”extensions; in two separate reactions, the average length of the ssDNA tails was either 30 or 70 nucleotides. When the partially single-strand dsDNA molecules were used as substrates in the strand transfer reaction, they were effectively paired with ssDNA to form joint molecules by exonuclease-deficient Rrpl-N551 (Fig. 7). In addition, a peak of activity corresponding to the elution position of Rrpl-N551 protein was observed when this substrate was used to assay fractions from the Bio-Rex 70 column shown in Fig. 6 (data not shown). Two other proteins with ssDNA renaturation activity were also tested in this assay. E. coli single-strand DNA-binding protein pairs these homologous molecules very inefficiently, whereas calf thymus histone H1 can form joint molecules (data not shown). In a separate experiment, linear dsDNA substrates with 3”protruding ssDNA tails were tested in a similar reaction. Rrpl-N551- catalyzed joint molecule formation was just as efficient with

2080 Recombinant Rrpl Protein Is Multifunctional TABLE I1

Quantitative analysis of strand transfer, 3'-exonuclease, and AP endonuclease activities Numbers in parentheses are standard deviations. Where standard deviation is shown, the values are the means of two to six determinations.

Protein' DNA strand transferb 3'-Exonucleasec AP endonucleased (unitslrnd X IO" unitslmg (unitslrnd X IO-'

Rrpl-Rev Fraction I ND' Rrpl-l-N551, Frac- ND 2 (2.0)

Rrpl-693 tion I1

Fraction I 1.4 (0.4) Fraction I1 6.7 (2.0) 300 (12) Fraction 111-HS 7.3 (2.2) 290 (25) 1.2 (0.3) Fraction 111-S-400 8.9 (1.5) 300 (65)

Rrpl-1-679, Frac- 8.7 (1.3) 80 (16) 1 .o Exonuclease 111 ND 51,000 (1100) 1.7 (0.4)

tion VI

'The protein fractions are defined as follows: Fraction I, 0.6 M NaCl extract; Fraction 11, Bio-Rex 70; Fraction 111-HS, heparin-Sepharose;

* One unit of DNA strand transfer activity converts >50% of the dsDNA substrate to product in 15 min at 37 "C. Fraction 111-S-400, Sephacryl S-400 step is substituted for the heparin-Sepharose step.

One unit of 3'-exonuclease releases 1 nmol of acid-soluble nucleotides in 30 min at 30 "C. One unit of AP endonuclease activity cleaves 1 Dmol in 1 min at 30 "C.

e ND, not detectable.

A

B

(racllon no. lia

t - """"

Orlgh- 1 " " . + + + + - - llDNAnnaIurallon

astivity

m-

115 - 97 - 66-

45 -

29-

+ Rrpl-NS51

FIG. 6. Bio-Rex 70 chromatography of Rrpl-N551. 0.6 M NaCl was used to extract cells expressing Rrpl-N551 as described under "Experimental Procedures." This sample was diluted with an equal volume of buffer S2 and applied to a 5.0-ml Bio-Rex 70 column. The column was washed with buffer S2 containing 0.3 M NaCl and eluted with a gradient from 0.3 to 1.0 M NaCI. Fractions were assayed for strand transfer, ssDNA renaturation, and 3'-exonuclease activities as described. A, elution patterns for strand transfer (O), ssDNA renaturation (O), and 3'-exonuclease (0) activities are shown; B, aliquots of the indicated fractions were analyzed on a 9% gel by SDS- PAGE. The mobilities of marker proteins (in kilodaltons) and the origin of the resolving gel are indicated.

5'-resected dsDNA as it was with 3"resected dsDNA (data not shown).

As shown previously, strand-separating polyacrylamide gels can be used to determine the amount of single-strand linear

product - substrate -

FIG. 7. Joint molecule formation by Rrpl-N551 requires partially single-strand dsDNA. Strand transfer reactions were carried out under standard conditions using one of three dsDNA substrates that have 5'-single-strand overhangs of different lengths. In the control reaction, a 540-bp 5'-"P-labeled DdeI restriction fragment with a three-nucleotide overhang was used (untreated dsDNA). The same fragment was treated briefly with exonuclease I11 and purified by phenol extraction and ethanol precipitation (3'- resected dsDNA). Samples were prepared with average overhang lengths of either =35 or -70 nucleotides. DNA substrate and enzyme additions (20 ng of Rrpl-N551 or 50 ng of Rrpl-693) are indicated.

DNA produced during the strand transfer reaction (9). This method was used to determine the proportion of two- and three-strand reaction products present in the experiment shown in Fig. 7. Both Rrpl-693 and Rrpl-N551 promote the formation of a mixture of two- and three-strand DNA species; in contrast, calf thymus histone H1 primarily promotes the formation of three-strand reaction products (data not shown).

Polarity of RrpI Strand Transfer Reaction-Earlier studies of strand transfer reactions have demonstrated that joint molecule formation involving linear DNA molecules displays a polar character (17, 18, 29). For E. coli recA protein, this polarity may reflect a preferential binding to one DNA end that results from a polar polymerization of the protein on ssDNA (30, 31). In the case of the yeast strand transfer protein, Sepl, the polarity of strand transfer is thought to reflect the polarity of an intrinsic 5'-exonuclease (32). The Rrpl strand transfer polarity was determined to establish if it is consistent with a role of the Rrpl exonuclease in initiation of the strand transfer reaction.

The substrates used in this experiment are diagramed in Fig. 8A. Plasmid pMS215 is a construct with an -5-kb DNA

Recombinant Rrpl Protein Is Multifunctional 2081 Homologous mad

B. Kpnl Sac1 Srna 1

3ihQmkwoohomolocrv

orlgln -

SS BSK(-) -

FIG. 8. Polarity of joint molecule formation by R r p l cor- relates with polarity of Rrpl exonuclease. Strand transfer reactions were carried out under standard buffer conditions. 20-pl reactions contained 200 ng of linear pMS215 dsDNA and 100 ng of circular Bluescript SK(-) ssDNA (ss BSK(-1). A , dsDNA substrates are shown schematically. The plasmid pMS215 was linearized with SacI, KpnI, or SmaI. The SacI and KpnI ends were converted to blunt ends with T4 DNA polymerase. Thin black lines and hatched boxes indicate the regions of the linear dsDNA that are homologous and that are not homologous to the ssDNA substrate, respectively. The polarity of the dsDNA end that can base-pair with either (-)-ssDNA or (+)-ssDNA is indicated. B, additions of either 0.30 pg of Rrpl or 5.0 pg of recA protein were as indicated. In reactions with recA protein, ATP was included at a concentration of 2.5 mM, and 200 ng of E. coli single-strand binding protein was added after addition of recA protein. Incubation was carried out for 15 min (Rrpl) or 30 min (no enzyme or recA) at 37 "C. Samples were analyzed by gel electrophoresis as described under "Experimental Procedures."

fragment inserted into the vector pBluescript SK(-). Diges- tion of pMS215 at either end of the insert produces linear dsDNA substrates that are homologous to the circular ssDNA substrate at either the 5'- or 3'-end of the complementary strand of the dsDNA. The other end is unable to base-pair with the ssDNA due to noncomplementarity. By digestion in the middle of the insert region, a dsDNA substrate that is nonhomologous to the ssDNA at both ends is formed. T o eliminate any difference in the structure of the termini of these fragments, the termini of the cleavage products were all converted to blunt structures. Strand transfer reactions were carried out using ssDNA of either the (-)- or (+)-polarity. Fig. 8B shows results for the experiment with both Rrpl-693 and E. coli recA protein using (-)-ssDNA. Rrpl forms joint molecules efficiently only if 5'-terminal homology is present; the opposite preference is seen for recA protein, in agreement

with the results of others (10, 17, 18). As predicted, the opposite pattern was observed when (+)"DNA was substituted for (-)-ssDNA (data not shown). This result suggests that Rrpl exonuclease acting at the 3'-end of the noncomple- mentary strand of the dsDNA reveals a region of ssDNA at the 5'-end of the complementary strand. This single-strand region can then anneal with the circular ssDNA during the formation of joint molecules. Data presented above (Fig. 7) indicate that such a tailed dsDNA molecule is a likely intermediate in the strand transfer reaction.

DISCUSSION

Drosophila Rrpl is a low abundance protein found in Dro- sophila embryos and in embryo-derived tissue culture cells. Therefore, preparation of homogeneous Rrpl from Drosophila tissues for enzymatic studies is difficult. To further charac- terize this protein, we have developed an E. coli expression system that allows efficient preparation of active Rrpl protein. We previously demonstrated that four enzymatic activities are associated with a highly purified Rrpl fraction obtained from Drosophila embryos. The work presented here provides strong evidence that the single Rrpl polypeptide is sufficient for all four activities: DNA strand transfer, ssDNA renaturation, dsDNA 3'-exonuclease, and AP endonuclease (Fig. 5 and Table 11). When purified from E. coli, the strand transfer-, 3'-exonuclease-, and AP endonuclease-specific activities are very similar for native and recombinant Rrpl proteins.

The COOH-terminal third of the Rrpl protein sequence is homologous to E. coli exonuclease 111. Analysis of a COOH- terminally deleted Rrpl protein, Rrpl-N551, establishes that the deleted region (amino acids 552-693) is necessary for both the Rrpl 3'-exonuclease function and the strand transfer function. The strand transfer activity of Rrpl-N551 can be restored if exonuclease activity (either 3'-exonuclease (Fig. 7) or 5'-exonuclease (data not shown)) is provided in trans by predigesting the dsDNA. This suggests a role for the exonuclease in initiation of the strand transfer reaction since it demonstrates utilization of a putative reaction intermediate by Rrpl (partially single-strand dsDNA). Analysis of the polarity of the Rrpl strand transfer reaction confirms this interpretation by also demonstrating a polar requirement for terminal homology that is consistent with the known polarity of Rrpl exonuclease (Fig. 8). Johnson and Kolodner (32) have suggested that yeast Sepl protein, a strand transfer protein with an intrinsic 5'-exonuclease, utilizes a mechanism that also requires a partially single-strand dsDNA intermediate (see discussion below).

This work suggests that a two-strand annealing reaction is likely to be involved in the phase of the strand transfer reaction in which base pairing is established between the ssDNA and dsDNA substrates to form a three-strand product. This work does not address any subsequent phase of the strand transfer reaction carried out by Rrpl; however, the fate of these three-strand molecules has been addressed in some experiments (data not shown) (9). In the presence of Rrpl protein, three-strand molecules resolve into two products by strand displacement (one linear ssDNA product and one dsDNA product) with a high efficiency if the displaced strand is <400 nucleotides long (9). This strand displacement reaction requires Rrpl action on three-strand joint molecules since Rrpl does not produce linear ssDNA from dsDNA alone and since other proteins such as histone H1 that renature homologous DNA strands are inefficient in this reaction (data not shown). Additional investigation will be required to char- acterize this putative second phase of the Rrpl-catalyzed strand transfer reaction. In particular, it remains unclear what

2082 Recombinant Rrpl Prote in Is Mult i funct ional

mechanism might govern a switch from one phase of the reaction to another.

NHz-terminally deleted forms of Rrpl have not yet been characterized to establish which regions of the sequence are required for strand transfer. However, it is shown here that the NHz-terminal region of Rrpl protein (amino acids 1-452) is sufficient to carry out ssDNA renaturation, suggesting that i t contains a ssDNA-binding domain. In previous enzymatic studies of Rrpl, it was suggested that the presence of a ssDNA-binding domain may account for some of the unique properties of this protein (22).

In another recombination system, the X red pathway, the involvement of coordinated exonuclease and ssDNA renaturation activities is also known. This system involves two ge- netically defined loci that encode X-exonuclease and a ssDNA renaturation protein known as P, respectively (3). Both functions are required for this recombination pathway, and the two proteins are thought to form a heteromeric complex with each other. However, i n uitro biochemical analysis of the X- exonuclease-P-protein complex is not extensive. Another well- characterized strand transfer protein, Sepl (32, 33), has been described that has intrinsic exonuclease and ssDNA renaturation activities. However, the similarity between Rrpl and Sepl is limited since their properties differ significantly in several respects. Rrpl is not required at near-stoichiometric amounts to carry out strand transfer as is Sepl (6). In contrast to the Rrpl exonuclease specificity, the exonuclease of Sepl is 5'-3'-specific and prefers ssDNA to dsDNA substrates. Furthermore, there is no similarity between the amino acid sequences of these two proteins. However, Rrpl and Sepl demonstrate a mechanistic similarity in the i n vitro strand transfer reaction they carry out. This common mechanism, which involves essential exonucleolytic processing of dsDNA ends prior to formation of joint molecules and initiation of strand transfer, has been established by biochemical analysis of these two eukaryotic proteins i n uitro. A similar mechanism is suggested for X red recombination.

Rrpl protein is an unusual member of a family of DNA repair proteins that is conserved from E. coli to man. (The members of the family are E. coli exonuclease 111, Streptococ- cus pneumoniae exonuclease A, Drosophila Rrpl (14), human APE (34), bovine BAP (35), and mouse APEX (36).) A large NHz-terminal region of 427 amino acids found in Rrpl is not present in the bacterial members of the family. The other eukaryotic members of the family have short NHz-terminal regions not homologous to the prokaryotic proteins that are -60 amino acids in length. These regions are not similar in sequence to Rrpl, but are highly conserved among the mouse, bovine, and human sequences (34-36). No functions associated with the NHz-terminal regions of the bovine or mouse proteins have been described. Possible nuclear localization signals are found in this region of the APE protein (34), which is known to be a nuclear protein.

Rrpl protein is an efficient AP endonuclease. The specific activities of Rrpl exonuclease and E. coli exonuclease I11 are comparable in the AP site cleavage assay. However, an inter- esting result presented here is that the ratio of AP endonuclease-specific activity to exonuclease-specific activity for these two related proteins is different by 2 orders of magnitude (Table 11). Preliminary studies indicate that Rrpl exonuclease demonstrates product inhibition and low processivity that

could contribute to low exonuclease a ~ t i v i t y . ~ Other members of the Rrpl protein family have been described that are completely deficient in exonuclease activity (34, 35).

The in vitro analysis of Rrpl protein presented here extends our understanding of its biochemical properties, but does not address the issue of its biological function i n uivo. The ability to express active recombinant Rrpl will allow us to address its function in heterologous systems in the near future. Since Drosophila strains mutated in Rrpl are not yet known, construction and characterization of such mutants are required to define Rrpl function in the homologous system.

Acknowledgments-M. S. thanks Bob Voelker, Mike Resnick, Tao Hsieh, and Ky Lowenhaupt for helpful comments on this manuscript and Burke Judd for continuing encouragement and support. We also thank Ky Lowenhaupt for preparing anti-Rrpl antiserum and gen- erously providing it to us.

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