From the Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware 19716
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ABSTRACT |
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It has been shown in vitro that
Saccharomyces cerevisiae strand exchange protein 1 (Sep1)
promotes the transfer of one strand of a linear duplex DNA to a
homologous single-stranded DNA circle. Sep1 also has an exonuclease
active on DNA and RNA. By using exonuclease III-treated linear duplex
DNA with various lengths of single-stranded tail as well as
Ca2+ to inhibit the exonuclease activity of Sep1, we show
that the processivity of exonuclease activity of Sep1 is greater
than previously reported. The results in this work also demonstrate
that the joint molecule between the linear duplex and single-stranded
circle observed from the Sep1-promoted strand transfer reaction is just the pairing between the long single-stranded tail of the linear duplex
DNA (generated by the exonuclease activity of Sep1) and the
single-stranded circular DNA. When a synthetic Holliday junction was
used as substrate, branch migration facilitated by Sep1 could not be
detected. Finally, using electron microscopy no -structure, a joint
molecule with displaced single-stranded DNA tail that indicates branch
migration could be observed. The results imply that Sep1 cannot promote
branch migration in vitro. Further investigation is needed
to determine the role of Sep1 in recombination in vivo.
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INTRODUCTION |
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The Sep1 (also known as Kem1, Xrn1, Rar5, DST2/Stp) protein of
Saccharomyces cerevisiae is a 175,000-kDa protein with
proposed roles in nuclear fusion (1), RNA turnover (2-4), and the
stability of replication-defective plasmids (5) as well as in
microtubular cytoskeleton functions (6), G4 DNA pairing (7) and DNA
recombination (8-10).
Most of our knowledge about homologous pairing and strand exchange
processes comes from the studies of Escherichia coli RecA protein. The RecA paradigm has wide generality. Analogues of RecA have
been isolated recently from a number of organisms, including bacteriophage T4 UvsX (11, 12), yeast Rad51 and Dmc1 (13, 14), as well
as human Rad51 (15). However, it has been suggested that Sep1 is a
member of a different class of homologous pairing proteins, the
E. coli RecT-RecE family (16). The characteristic feature of
this family is that an exonuclease first exposes complementary single
strands, a plectonemic joint is formed in the absence of a high-energy
cofactor (ATP), and strand exchange is subsequently promoted. RecT-RecE
homologs, like RecA, are widespread in nature and include the
bacteriophage l, and
proteins (17).
Sep1, like RecA of E. coli, has been shown to promote the transfer of one strand of a linear duplex DNA to a homologous single-stranded DNA circle (18, 19). Sep1 not only promotes this type of strand transfer, but also performs the renaturation of homologous single-stranded DNA molecules (20). Furthermore, it contains an intrinsic 5' to 3' exonuclease activity, which exposes a terminal single-stranded region in linear duplex DNA. It has been shown that the exonuclease activity of Sep1 is slow and nonprocessive, with a turnover number of 20 nucleotides/min and a processivity of ~45 nucleotides on duplex DNA (21, 22). It was shown that for Sep1 to initiate strand exchange, the linear duplex must have a single-stranded tail greater than 20 nucleotides in length; this single-stranded tail can be produced by Sep1 or by an exogenous exonuclease (21, 22). Once the small single-stranded DNA tail has been produced, Sep1 nuclease activity is dispensable for the strand displacement and branch migration phase of the strand exchange reaction (21, 22).
The assumption that Sep1 is involved in DNA recombination came from genetic analysis of Sep1 mutants (9, 10, 23) and the in vitro strand exchange activity of the purified Sep1 protein mentioned above (18, 24-26). It was proposed that in the presence of Mg2+ or Ca2+ (when single-stranded tails are provided), Sep1 could promote a strand-exchange reaction, replace one strand in the double-stranded DNA with the homologous single-stranded circular DNA, to form a nicked double-stranded circle and a single-stranded linear molecule (21, 22).
However, it is yet to be demonstrated that Sep1 has a direct role in
recombination in S. cerevisiae. In fact, Sep1 has several biochemical activities in vitro that seem to have little to
do with recombination (6, 7, 27). In addition, Sep1 mutants do not
have a strong Rec phenotype and the results are
strikingly assay dependent (24). Furthermore, sep1 gene has
not been picked up in general screens for yeast recombination mutants
(e.g. Ref. 28).
In this work, the standard strand transfer assay (the transfer of
single-stranded DNA from a linear duplex to circular ssDNA) was
re-examined using exonuclease III-generated linear duplex molecules
with various lengths of single-stranded tails, in combination with the
presence of Ca2+, to inhibit the exonuclease activity of
Sep1. The results show that the processivity of the exonuclease
activity of Sep1 is much greater than previously reported. The data
also indicate that the joint molecule apparently formed between the
linear duplex and the single-stranded circle observed from the
Sep-promoted strand transfer reaction is just the pairing between the
long single-stranded tail of the linear duplex and the single-stranded circle. Furthermore, with a synthetic Holliday junction as substrate, we cannot detect any branch migration promoted by Sep1. In addition, electron microscopic studies failed to show the so-called
-structure, an intermediate joint molecule with a displaced ssDNA
tail, a characteristic indicator that DNA strand exchange had occurred. We conclude that Sep1 cannot promote branch migration in
vitro. Therefore, the main biochemical functions of Sep1 appear to
be its exonuclease activity and its function as an aggregating agent. Further investigation is needed to determine the role of Sep1 in
recombination.
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EXPERIMENTAL PROCEDURES |
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Strains-- E. coli Xl1-Blue and helper phage VCSM13 were from Stratagene Cloning Systems. S. cerevisiae strain BJ5464/pRDK249 was generous gift from Dr. Richard D. Kolodner of Dana-Farber Cancer Institute.
Enzymes and Chemicals-- All restriction endonucleases were from Promega. T4 DNA ligase, T4 polynucleotide kinase, and shrimp alkaline phosphatase were from New England Biolabs. DE52 was from Whatman Inc. PBE94 and Sephacryl S-200 were from Pharmacia Biotech Inc. Benzamidine hydrochloride, leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, and dithiothreitol were from Sigma.
DNA Substrates-- Phagemid pBluescript II KS+ (hereafter called KS+) was from Stratagene Cloning Systems. The double-stranded pBluescript KS+ supercoiled DNA was purified according to a standard procedure (29) and linearized by digestion with XhoI (called KS+/XhoI hereafter) or by EcoRV (called KS+/EcoRV hereafter). Single-stranded DNA was purified from Xl1-Blue/KS+ infected by the helper phage VCSM13 according the manufacture's manual.
Protein Purifications--
Sep1 was purified from yeast strain
BJ5464/pRDK249 following a published method (19) with the following
modifications. The cells were broken using a French Press at 1,500 p.s.i. followed by centrifugation at 10,000 rpm for 30 min in a Sovall
SS34 rotor. The pellet was resuspended and fractionated by Polymin P
precipitation and ammonium sulfate precipitation. Redissolved proteins
were purified using a DE52 column (2.5 cm2 × 100 cm), a
single-stranded DNA cellulose column (1.8 cm2 × 11 cm), a
PBE 94 column (0.38 cm2 × 9.8 cm), a Sephacryl S-200
column (2 cm2 × 68 cm), and a Mono S column (Pharmacia HR
5/5). The final fraction was dialyzed against storage buffer (20 mM Tris, pH 7.5, 10 mM -mercaptoethanol, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 100 mM NaCl, and 60% glycerol overnight) and stored at
80 °C. The step of concentrating Sep1 by dialysis against 20% PEG
80001 in the published method
(19) was omitted, because we found that PEG could interfere with the
strand exchange assay. The Sep1 protein was >99% pure as judged by
5% SDS-polyacrylamide gel electrophoresis.
Assay of Strand Exchange Reactions by Sep1-- In a reaction volume of 30 µl, 0.05 µg of single-strand circular pBluescript KS+ DNA (5 µM) and 0.1 µg (5 µM) of linear double-stranded pBluescript KS+ DNA were incubated either with 1 µg of Sep1 (0.19 µM) in a Mg2+ buffer containing 33 mM Tris-HCl, pH 7.5, 15 mM MgCl2, 2 mM dithiothreitol, and 100 µg/ml bovine serum albumin, or in Ca2+ buffer containing 33 mM Tris-HCl, pH 7.5, 5 mM CaCl2, 2 mM dithiothreitol, 100 µg/ml bovine serum albumin at 30 °C for 30 min or as indicated. The reaction was stopped by adding 2.5 × stop solution containing 15% glycerol, 5% SDS, and 0.125% bromphenol blue. The product was analyzed by running on a 0.8% agarose gel in 0.5 × TBE buffer.
Construction of Holliday Junctions-- The Holliday Junction structure was constructed following Panyutin and Hsieh (Ref. 30; see Fig. 4A) with the following modifications. Plasmid pBluescript KS+ was digested with NaeI and EcoRI. The 377-base pair fragment was purified from low-melting agarose gel electrophoresis and used as the homologous arms in the junction structure. The synthetic oligonucleotides 5'-ACCATGCTCGAGATTACGAGATATCGATGCATGCG-3' and 5'-AATTCGCATGCATCGATATAATACGTGAGGCCTAGGATC-3' were annealed and ligated to the 377-base pair arm, and formed the partial duplex substrate S1 for the Holliday junction. The synthetic oligonucleotides 5'-GATCCTAGGCCTCACGTATTATATCGATGCATGCG-3' and 5'-AATTCGCATGCATCGATATCTCGTAATCTCGAGCATGGT-3' were annealed and ligated to the 377-base pair to form S2. These partial duplex substrates S1 and S2 were then purified from 6% native acrylamide gel. S2 was then labeled at its 5'-end with [32P]ATP by T4 polynucleotides kinase.
Assay for Branch Migration with the Synthesized Holliday Junction-- The 32P-labeled S2 described above was mixed with a 10-fold molar excess of S1 (unlabeled) in different reaction buffers as indicated in the text in a total volume of 10 µl. After incubation at an appropriate temperature, the reaction was stopped by 10 µl of ice-cold reaction buffer containing 1 µg/ml ethidium bromide and kept on ice prior to electrophoresis. The branch migration products of Holliday junctions were analyzed by electrophoresis in 4% native polyacrylamide gel in 1 × TAE buffer containing 1 µg/ml ethidium bromide and 10 mM MgCl2 or 5 mM CaCl2 at 5 V/cm for 10 h at 4 °C. Gels were dried, autoradiographed, and quantified using a Bio-Rad Molecular Analysis System.
Electron Microscopy-- The DNA samples were prepared for microscopy using a method by Pe'rez-Morga and Englund (31) which is modified from Davis et al. (32). Samples on Parlodion-coated copper grids were stained with uranyl acetate, rotary shadowed with Pt/Pd, and viewed in a Zeiss CEM 902 electron microscope.
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RESULTS |
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Properties of the Purified Sep1 Protein-- Fig. 1A shows that purified Sep1 has an intrinsic exonuclease activity in the presence of Mg2+ buffer; this exonuclease activity is strongly inhibited by Ca2+ (19). It has also been shown that Sep1 can promote joint molecule formation and subsequently strand transfer between single-stranded circular DNA and double linear DNA in the presence of Mg2+ (diagrammed on top of Fig. 1B). However, if Ca2+ instead of Mg2+ buffer is used, then no joint molecule can be detected (19). Using a 3-kb linear duplex DNA and its homologous single-stranded circular DNA as substrates, we were able to reproduce those published results. As shown in Fig. 1B, Sep1 promotes joint molecule formation between single-stranded circular DNA and double-stranded linear DNA in the presence of Mg2+, regardless of whether the duplex DNA has sticky ends or blunt ends, but not in Ca2+. Note that none of the joint molecules formed from the Mg2+ reactions are the complete strand transfer reaction products, nicked circular duplex DNA, despite the long reaction time. Fig. 1C shows that if the duplex DNA was first treated with exonuclease III to produce single-stranded tails then the joint molecule could be formed in the presence of Ca2+ buffer. Again, none of the joint molecules formed from the Ca2+ reactions are the complete strand transfer reaction products (nicked duplex circle). These results confirm the earlier conclusions that a limited exonuclease digestion from the ends of the double-stranded DNA is required to initiate the strand-exchange reaction promoted by Sep1 (21, 22). Also, if the intrinsic exonuclease activity of Sep1 is inhibited, then for Sep1 to initiate strand transfer reaction, the double-stranded DNA molecules have to have single-stranded tails that are homologous to the circular single-stranded DNA (21, 22).
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In Mg2+ Buffer, Sep1 Can Promote the Formation of Joint Molecules, but the Joint Molecules Cannot Branch Migrate to Form Complete Strand Transfer Products-- As mentioned above, in the reactions shown in Fig. 1, the substrates used were pBluescript phagemid (3 kb) which are much smaller than generally used in this type of reaction (M13, 8 kb). Under these conditions, the Sep1-mediated strand exchange reaction does not go to completion within 90 min. However, the same reaction performed by E. coli protein RecA could be completed in less than 30 min (data not shown). To investigate whether Sep1 can drive the reaction to completion if enough time is given, we decided to repeat the Sep1-mediated strand transfer reaction with Mg2+ buffer. However, in this experiment we monitored the reaction during a period of 3[frax,1,2] h. As shown in Fig. 2, in the presence of Mg2+, we detected increasing amounts of joint molecules between 10 and 120 min of reaction time (lanes 2-10 in Fig. 2). In agarose gels, the joint molecules formed during this period of time migrate slower than the nicked duplex circle (lane 17 in Fig. 2) that one would expect to see if the strand transfer were completed. The joint molecules in this group also show increasing mobility with increasing reaction time (lanes 2-10). The samples taken after 120 min of reaction (from 135 to 210 min; lanes 11-16) indicate surprisingly that the joint molecules migrate much faster than the nicked duplex circle (lane 17).
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Joint Molecules Form Without Branch Migration in Ca2+
Buffer, if the Duplex DNA Is First Treated with Exonuclease III to
Produce Single-stranded Tails--
The results shown in Fig. 2
indicate that the exonuclease activity of Sep1 is processive in the
presence of Mg2+ and does not promote branch migration. It
has been shown that when provided with 20-40 nucleotides of the
single-stranded region at the ends of the linear duplex DNA, Sep1 can
promote branch migration if its exonuclease activity is inhibited by
Ca2+ (21, 22). To test this directly, we first digested the
double-stranded DNA substrate with exonuclease III to produce
single-stranded tails (the polarity of these tails are opposite to the
tails produced by Sep1). The Sep1-mediated strand transfer reaction in
the presence of Ca2+ was then performed. If Sep1 could
promote branch migration under these conditions, then one should expect
to see the fully exchanged circular duplex DNA with a small gap which
should migrate a little faster than the nicked circular DNA on the gel.
On the other hand, if Sep1 could promote only the reannealing of the
complementary single-stranded regions from the resected duplex DNA and
the circular ssDNA, then the joint molecules formed (-structures)
should move slower than nicked circular DNA in agarose gels. The lower
mobility would result from the protruded linear duplex DNA tail in the joint molecule. However, if the linear double-stranded DNA substrate is
overdigested with exonuclease III, the joint molecules formed will be a
circular DNA with a large gap, which should move much faster than the
nicked circular DNA. The results are shown in Fig.
3A. The Sep1-mediated strand
transfer reaction between exonuclease III-resected linear dsDNAs
(pBluescript) with various lengths of single-stranded tails on the
linear duplex DNAs and its homologous circular ssDNA was performed in
Ca2+ buffers for 150 min. Even after such long reaction
times, the products of the performed strand transfer reactions are
joint molecules that either migrate slower (
-structures, lanes
6-11) or faster (gapped circular, lane 12) than nicked
circular DNA (lane 2).
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Exonuclease III-resected Double-stranded DNA with a Single-stranded Tail Can Also Form Joint Molecules Spontaneously with Higher Concentrations of Substrate-- Fig. 3A shows again that Sep1 does not promote branch migration, and the joint molecules observed here are formed from reannealing of the single-stranded regions of two substrate molecules. To confirm this conclusion, we decided to compare the joint molecule formed from exonuclease III-resected double-stranded DNA and single-stranded circular DNA using either Sep1 in Ca2+ buffer or spontaneous reannealing in the same buffer. The results are shown in Fig. 3B. Lanes 4-8 show the results where the double-stranded DNA were first resected with exonuclease III for increasing periods of time, and then used as substrates. The reactions were carried out with the resected linear duplex DNA plus circular ssDNA and Sep1 for 90 min in Ca 2+ buffer. Lanes 9-13 show the reactions of the same substrates as in lanes 4-8 but approximately 4-fold more concentrated without the addition of Sep1. Clearly both sets of experiments produce exactly the same products, with or without Sep1. We conclude again that Sep1 does not promote branch migration. Note that higher concentrations of the substrates have to be used for the spontaneous reannealing reactions to produce the same joint molecules as those promoted by Sep1. This is not surprising, since Sep1 aggregates DNA, thus facilitating the reannealing process (20).
Sep1 Does Not Promote Branch Migration Using a Synthetic Holliday Junction as Substrate-- To test further the ability of Sep1 to promote branch migration, we used a synthetic Holliday junction (30) to test directly whether Sep1 can promote branch migration on this complex. The synthetic Holliday junction as shown in Fig. 4A, was constructed by annealing two homologous duplex DNAs each having two single-stranded tails at one end that are complementary to the corresponding single-stranded tails of the other duplex. Upon annealing of the two duplex DNAs, a four-stranded complex (Holliday junction) is formed. In this complex, branch migration can only proceed to the opposite end of the duplexes (only at this direction is there sequence homology between the two original duplexes). The ultimate product of branch migration in this system is complete strand exchange and the formation of two duplex DNA products. Using this system, the rate of branch migration can be determined by the appearance of the product duplex which migrate much faster than the four-stranded complex (Holliday junction) during gel electrophoresis. The top two panels of Fig. 4B show the spontaneous branch migration of the synthetic Holliday junction in Mg2+ and Ca2+ at 50 °C. It is clear that the duplex products are evident after ~60 min in Mg2+, and after ~30 min in Ca2+. The smears at the tops of these two gels indicate the Holliday junctions at different stages of branch migration. The spontaneous branch migration rates measured from these two experiments agree with the published results using a similar synthetic Holliday junction (30). Therefore, we are confident that we have constructed the proper synthetic Holliday junction (30). The third panel of Fig. 4B is the result of spontaneous branch migration of the synthetic Holliday junction in Ca2+ at 30 °C, which is the condition for Sep1-promoted strand transfer reaction. Without any protein, the spontaneous branch migration at this temperature in Ca2+ is so slow that no products can be detected after 5 h. If Sep1 could promote significant branch migration, some duplex products should be observed under these conditions. The bottom panel of Fig. 4B shows no detectable duplex products in the presence Sep1. Except on the top of this gel, the bands corresponding to the synthetic Holliday junctions show a small degree of smearing, indicating that there is a little bit of branch migration, but not enough to produce the products. Note that the duplex DNAs used to construct this synthetic Holliday junction are only about 400 base pairs.
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Electron Microscopy of Strand Transfer Products Revealed No
Evidence of Joint Molecules with a Displaced Single-stranded
DNA--
Finally, we have examined the products of the Sep1-promoted
strand transfer reaction directly by electron microscopy. Portions of
reactions used in agarose gel analysis shown in Fig. 2 were analyzed
directly by electron microscopy (as described under "Experimental Procedures") without being deproteinized first. Branch migration should lead to joint molecules containing an -structure, where one
of the DNA strands from the duplex was displaced by the circular single-stranded DNA. On the other hand, if Sep1 cannot promote branch
migration, then one should expect to see only
-structures, where
there is no displaced single-stranded tail in the joint molecule. The
products of strand transfer reactions promoted by Sep1 in the presence
of Mg2+ were analyzed by EM (as shown in Fig.
5). Panels A-I, are
representative micrographs taken from samples of increasing reaction
times as those shown, respectively, in lanes 4 (A
and B, 30 min), 8 (C and D,
90 min), 10 (E and F, 120 min),
12 (G, 150 min), 14 (H, 180 min), and 16 (I, 210 min) of Fig. 2. Out of ~20
joint molecules scored from each sample, we could only detect
-structures, no other types of joint molecules were observed. The
electron micrographs also show that joint molecules of samples from
longer reaction times have shorter tails than those from shorter
reaction times. The EM results confirm the interpretations of the
agarose gel shown in Fig. 2, indicating that the intrinsic exonuclease
activity of Sep1 in Mg2+ is processive and that Sep1 cannot
promote branch migration.
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DISCUSSION |
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Unlike RecA of E. coli, we have found that Sep1 does
not promote complete strand transfer reactions in vitro; the
assay is a model strand exchange reaction, in which a single-stranded
circular DNA (pBluescript, 3 kb) and its homologous linear duplex DNA
were used as substrates. The joint molecules observed appear to be just
the annealing of the single-stranded tail of the duplex DNA (generated
by the intrinsic exonuclease activity of Sep1), and its complementary
bases on the single-stranded circular DNA. As shown in Fig.
6, we propose that Sep1 initially digests
the double-stranded DNA in 5' 3' direction and produces two
single-stranded DNA tails. The complementary tail then anneals with the
single-stranded circular DNA and produces an initial joint molecule.
Sep1 continuously hydrolyses both ends of the linear duplex DNA in the
joint molecule and produces longer single-stranded tails. The joint
region between the single-stranded circular DNA and the complementary
single-stranded tail gets longer, while the double-stranded region in
the linear duplex DNA gets shorter. As a consequence, the joint
molecule moves faster and faster on agarose gels but still moves slower than double-stranded nicked circular DNA. When the linear DNA is
digested to half of its original length, the single-stranded tail on
the joint molecule is released, yielding a gapped double-stranded circular DNA, which moves faster than the double-stranded nicked circular DNA. The gapped duplex DNA is continuously digested by Sep1
resulting in only single-stranded circular DNA in the reaction mixture.
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This model strand exchange reaction has been used extensively for in vitro studies on recombination of DNA molecules, especially for reactions promoted by RecA protein; the reaction with RecA is presumably preceded by a homology search and pairing process. The results shown in this work suggest that detection of joint molecules or even strand exchange in vitro promoted by a protein does not necessarily imply that this particular protein is capable of seeking homology and promoting branch migration. This is especially important for those proteins, such as Sep1, that have an intrinsic exonuclease activity.
In many studies concerning the strand exchange proteins, volume-occupying agents such as PEG are added to enhance the strand exchange reaction, presumably to increase the local concentration of substrate DNA (26, 33, 34). PEG has also often been used to concentrate protein during the final stages of protein purification, including the published protocol for purification of Sep1 (19). We have found that even without any protein, PEG 8000 alone promotes strand exchange, provided the linear duplex DNA is first treated with exonuclease to produce single-stranded tails.2 It has also been reported that using systems similar to those used here, DNA strand-exchange reactions can be promoted in protein-free solutions under mild conditions with PEG, high concentrations of salt, or by proteins without any known roles in genetic recombination (35). These results indicate that in the presence of volume-occupying agents such as PEG, or proteins that can aggregate DNA, any protein which could produce single-stranded tails on duplex DNA would appear to promote strand exchange in this type of in vitro assay system. Sep1 has an intrinsic exonuclease activity and can aggregate DNA, therefore it can promote the formation of joint molecules without branch migration.
Previously we have shown that Sep1 can promote the formation of paranemic joints between two homologous DNA molecules (36). In those experiments we excluded the possibility that the exonuclease activity of Sep1 exposes complementary single-stranded regions that constitute the joint. Since Sep1 can aggregate DNA molecules, it is possible that Sep1 can facilitate homology searching by increasing the local concentration of DNA molecules and providing the single-stranded tail for proteins such as Rad 51 (a RecA homolog of S. cerevisiae) to perform the actual homology search and subsequent branch migration. Hence, although the results here show that Sep1 cannot promote branch migration in vitro, we still cannot exclude the possibility that Sep1 could be indirectly involved in recombination in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. Kirk J. Czymmek of biological electron microscopy facility of the University of Delaware for help with the electron microscopy. We thank Drs. Nick Cozzarelli, Roberta Colman, Mahendra Jain, Ned Seeman, and Colin Thorpe for comments on the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Biotechnology, Sichun Union University,
People's Republic of China.
§ Supported by a grant from the Howard Hughes Medical Institute through the Undergraduate Biological Science Education Program.
¶ To whom correspondence should be addressed.
1 The abbreviations used are: PEG, polyethylene glycol; kb, kilobase(s); ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.
2 Z. Zhang and J. Chen, manuscript in preparation.
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REFERENCES |
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