Hereditas 1 17: 1-9 (1992)

RecA-like strand-transfer activity at the meiotic prophase in Bombyx mori BENTE WISCHMANN Department of Physiology, Carlsberg Laboratory, Copenhagen, Denmark

WISCHMANN,B. 1992. RecA-like strand-transfer activity at the meiotic prophase in Bombyx mori. Hereditas 1 1 7 1-9. Lund, Sweden. ISSN 0018-0661. Received July 08, 1991. Accepted February 28, 1992 An ATP-independent strand-transfer activity has been identified in nuclear extracts prepared from Drosophila tissue culture cells and isolated nuclei from Bombyx testes. Extraction of the activity from testes at larval stages where the majority of the cells were in meiotic prophase was only possible when the chromosome scaffold/synaptonemal complex was dissolved by addition of high concentrations of DTT (80 mM). No cross reaction was detected when partly purified extracts were assayed with antibodies against E. coli RecA protein. Bente Wischmann, Department of Physiology, Carlsberg Laboratory, GI. Carlsberg Vej 10, DK-Z~IN Copenhagen, Denmark

The key event of meiosis in normal diploid eukaryotes, on which both the genetic recombination and a proper segregation depend, is the precise pairing of homologous chromosomes at the meiotic prophase. Chromosome synapsis is accompanied in virtually all organisms by formation of a synaptonemal complex between the homologous chromosomes. The assembly of the synaptonemal complex at zygotene and its appearance and behavior in pachytene nuclei have been the subject of a large number of ultrastructural studies (JOHN 1990). The mechanism responsible for the initial recognition of homologues preceding pairing and synaptonemal complex formation is less well characterized at the ultrastructural level and not at all biochemically. It is a common notion that temporary homologous pairing of complementary DNA sequences yielding stretches of heteroduplex DNA is an important step in recombination of linked genes (JOHN 1990 and references cited herein). Less discussed but equally likely is the involvement of transient homologous pairing at the DNA level in the initial recognition between homologous chromosomes prior to synapsis and synaptonemal complex formation (SMITHIESand POWERS1986; CARPENTER1987; STERN and HOTTA 1987). Proteins which catalyze homologous pairing of complementary DNA molecules by a strand transfer reaction - RecA-like proteins -are thus among the possible candidates for proteins with specific functions at the meiotic prophase.

In this report an activity is described in partially purified extracts from testes of the silkworm, Bombyx mori, that catalyzes the transfer of one strand of a double-stranded (ds) DNA molecule to its complementary sequence on a single-stranded (ss) circular DNA molecule.

Materials and methods Cell lines

The RecA-protein was isolated from E. coli strain DR1453 containing multiple copies of the plasmid DR1453, a derivative of PBR322 in which the srl-RecA region from E. coli is inserted. The plasmid was kindly provided by C. M. Radding, Yale School of Medicine, USA. The Drosophifa melanogaster cell line KcO was grown at 22°C in Ekhaliers D22 tissue culture medium in 1OOOml flasks with aeration and magnetic stirring. The silkworms, Bombyx mori, were maintained as a randomly breeding population on an artificial mulberry diet (Silkmate E, Nihon Nosan Kogyo KK, Japan). Purification of the E. coli RecA-protein

The growth of the cells and the initial steps of the purification were essentially as described by SHIBATA et al. (1983). RecA-protein synthesis was induced by addition of nalidixic acid. Cell free

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extracts were obtained by treating the cells with lysozyme and the detergent Brij 58. After removal of cellular debris the nucleic acids were precipitated by polymin P. The pellet was washed in 0.5 M NaCl and extracted with 1.0 M NaCl. The extracted proteins were precipitated with 50 YOammoniumsutphate and dissolved in 1 M NaCl in 50mM Tris.HC1, pH 7.5, 1 mM EDTA, 10mM 8-mercaptoethanol, 10 YO glycerol. The protein fraction was desalted on a Sephadex G50 column, equilibrated with 20mM KPi, pH 6.8, 1 mM EDTA, 10 mM 8-mercaptoethanol, 10 % glycerol. The protein peak was directly loaded on a DEAEcellulose ion exchange column (DE-52, Whatman). The column was eluted with an ascending KCI gradient (0-0.5 M). Fractions containing ssDNA dependent ATPase activity ( WEINSTOCKet al. 1979) were collected, pooled and passed through a Sephacryl S-300 column previously equilibrated with 1 M NaCl in 50 mM Tris.HC1, pH 7.5, 1 mM EDTA, 10 mM b-mercaptoethanol, 10 YOglycerol. The active fractions were pooled. SDS-polyacrylamide gel electrophoresis of the isolated protein revealed, after silver staining, one prominent band with an apparent molecular weight of 38 kDa. Polyclonal antibodies were elicited in rabbits as described by HARBOEand INGILD (1983). The immunoglobulins were isolated from the serum on a protein-A Sepharose column (Pharmacia). Immunoblotting against cell free protein extracts of E. coli DR1453 identified a single band with a molecular weight of 38 kDa. Preparation of protein extracts

Kd) cells were harvested three times a week at a cell density of lo7 cells/ml and stored at -8O'C in growth medium. Testes from the silkworm B0rnby.x mori were dissected out, collected in PBS, immediately frozen in liquid N2 and kept at -80'C. The procedure for extracting nuclear proteins was the same for both cell types. The cells were disrupted in a Dounce homogenizer in HO-buffer ( 15 m M Tris.HC1, pH 7.9, 5 mM KCl, 0.5 mM MgCI?, 0.05 mM EDTA, 1 .O mM PMSF, 0.5 mM DTT, 0.05 OO/ Triton X-100)with a loose pestle, centrifuged at 4500 g for I0 min at 4'C. The pellet was resuspended in HO buffer and further homogenized with a tight pestle. The nuclei were collected by centrifugation (2500 g for 20 min) through a cushion of 0.8 M sucrose in HO buffer, resuspended in extraction buffer (EX: 30 mM Tris.HC1, pH 7.9, 1 .O mM EDTA, 350 mM NaCI, 10 YOglyc-

erol, 1 mM PMSF, 0.5-1.0mM DTT) by a few strokes with a loose pestle and left on ice for 60 min with occasional stirring. Following centrifugation (16,OOO g for 15 min), the nuclear extract was saved. Where indicated, the extraction was repeated with EX buffer containing 80mM DTT. The nucleic acids were precipitated by addition oft polymin P to a final concentration of 10 pl/ml. The nuclear proteins were fractionated by ammoniumsulphate precipitation. The 40-80 YO ammoniumsulphate cut was collected, resuspended in hydroxylapatite buffer (HA: 15 mM NaPi, pH 7.1, 10 YO glycerol, 0.01 % Triton X-100, 0.5 mM DTT), filtered through a 0.45 pm Millipore filter, and chromatographed on a hydroxylapatite column (Bio Rad HTP), equilibrated with HA buffer containing 200 mM potassium phosphate. Bound proteins were eluf-ed with 600mM potassium phosphate and dialyzed against HA buffer containing 100 mM NaCI. Protein concentrations were determined with a Bio Rad protein assay kit relative to bovine gamma globulin. The strand-transfer assay

M 13mp19 plasmid DNA was digested with restriction endonuclease DraI (Boehringer Mannheim) creating fragments of 4150, 2162, 365, 290, and 283 bp. The fragments were separated on a 1 % agarose gel and the three small fragments were excised, purified by electroelution, phenol extracted, ethanol precipitated, dried at room temperature, and stored in TE-buffer at 4°C. The 5' ends of the short dsDNA fragments were y3*Plabeled by use of a combined phosphatase-kinase reaction (BERGERand KIMMEL1987). The incorporation activity was measured by TCA (10 YO) precipitation and the total amount of incorporation per p g DNA was calculated to check the efficiency of the labeling reaction. The fragments were purified through a Sephadex G-100 gel filtration column or a prepacked Sephadex G-50 column. The standard reaction mixture for the strand-transfer assay contained 40 ng circular single-stranded (css) M13mp19 phage DNA, 0.750.90 ng of the labeled double-stranded (ds) DNA fragments, 12.5 mM MgCl,, 1 mM ATP, 18 mM creatinephosphate, 60 units/ml creatine kinase, and 530 pg/ml BSA (HSIEHet al. 1986). The reactions were initiated by addition of RecA or protein extracts, incubated at 3 0 T for 30min and terminated by addition of 5 YOSDS and 40 mM EDTA. After proteinase K treatment, the reaction prod-

STRAND-TRANSFER ACTIVITY IN BOMBYX MOM

Hereditas 117 (19%)

ucts were separated on 1.5 % agarose gel for 30 min at 10 V/cm. The agarose gel was dried onto filter paper and autoradiographed at -80°C with an intensifying screen. Quantification of band density was carried out by transmission measurements using a Shimadzu, Dual wavelength TLC scanner. For each lane of the gel, the band density was expressed as percentage of total density of the lane. SDS-PAGE, protein transfer and immunoblotting

The proteins were separated on 8 YO SDS-polyacrylamide gels at 110 V for 17 h, stained with coomassie brilliant blue and dried between cellophane sheets. For immunoblotting the proteins were electrotransferred to 0.45 pm nitrocellulose membranes. The filters were blocked with 1 % powdered skim milk in PBS for 30 min at 37°C and incubated in the primary antibody in PBS (17 pg IgG/ml) for 2 h at 37"C, washed five times in PBST (PBS, 0.05 YOtween 20) for 30 min at room temperature and incubated in peroxidase conjugated swine-antirabbit antibody (2 pl/ml) (Dacopatts, Copenhagen) for 1-2 h at 37°C. Following five washes in PBST for 30min, the color was developed. When L251-goatantirabbit IgG was used as the secondary antibody, the filters were incubated for 2 h at room temperature, washed in PBST, and autoradiographed at - 80°C with an intensifying screen.

Results The strand-transfer

assay

The DNA substrate used in the strand-transfer assay was circular single (+)strands (7.25 kb) from the M13mp19 phage and a mixture of short double-stranded restriction fragments (283, 290, and 365 bp) of the replicative form of M13mp19 labeled at both 5' ends with 32P.The preparation of the ~ ~ ~ P - l a b e lDraI e d fragments was analyzed for possible contamination of labeled singlestranded DNA molecules. The three DraI fragments eluted from the gel filtration column were separated on a 5 YO non-denaturing polyacrylamide gel. This resulted in four bands: two faster migrating bands representing the 365 bp fragment and a double band representing the comigrating two smaller fragments of 283 and 290 bp. In addition the gel revealed two more slowly migrating bands, the upper one of approximately half the

3

density of the lower band. Heat denatured labeled dsDNA showed the same mobility as the upper two bands, while S1 nuclease digestion, under conditions where S1 digests dsDNA, degraded all four bands to a single faster migrating band. These observations indicated that the labeled dsDNA was partly protected from digestion by proteins stemming from the labeling reactions (i.e., calf intestine phosphatase and/or T4 polynucleotide kinase), the upper two bands representing DraI fragments with bound protein. This was verified by excessive incubation of the DNA with proteinase K (400 pg/ml at 56°C for 1 h) after which the labeled dsDNA showed the expected three bands (one single and one double band) on 5 % non-denaturing p l y acrylamide gels. It was thus shown that the preparation of "P-labeled dsDNA was free of ssDNA, but that the insufficient proteinase K treatment following the labeling procedure resulted in labeled dsDNA with bound proteins. The product of the strand-transfer reaction (Fig. 1) catalyzed by RecA or RecA-like proteins was identified after proteinase K treatment and agarose gel electrophoresis as a band comigrating with the circular MI3 (+)strands (Fig. 5, lane 2), the lower band representing released short ( +)strands and excess labeled dsDNA. Nuclear extracts of somatic Drosophila and the meiotic Bombyx cells fractionated by ammonium sulphate precipitation were assayed in the presence and absence of ATP. Those experiments demonstrated (not shown) an ATP independency, i.e., omission of ATP and of the ATP-regenerating system produced a single radioactive band at the position of the dsDNA fragments. The same result was obtained if the reaction products were heat-denatured prior to electrophoresis. Finally, it was shown that ligase treatment of the labeled dsDNA fragments prior to the transfer reaction yields a band pattern clearly different from that produced by unligated dsDNA. The sensitivity of the transfer assay was assessed by maintaining a constant amount of cssM13 DNA (40ng) and increasing the amount of RecAprotein from 0.25 to 2.5 pg. The relative amount of label at the position of cssM13 DNA, signifying transfer of the short labeled (-)strands, increases sharply when the amount of RecA-protein is increased from 0.25 p g to approximately 0.9- 1.O pg per reaction (Fig. 2). Further addition of RecAprotein only produced a marginal additional increase in strand-transfer. Assuming that short labeled dsDNA is present in excess, this implies that about 1 pg RecA-protein is sufficient to com-

4 B. WlSCHMANN

Hereditas 117 (1992)

60-

40 -

20 -

-.

M

.c

. . 05

10

15

2.0

2.5

, l l g RecA

W 1

Fig. 1. Diagram of the strand-transfer reaction. The substrates are circular (+)strands of phage M13mp19 and three short (283. 290, and 365 bp) double-stranded DraI restriction fragments 32P-labeled at both 5’ ends which are homologous to segments of M13mp19. The reaction products are the 3’P-labeled ( -)strands completely or partially annealed to the circular ssDNA. In the case of stable partial transfer, labeled (+)strands contribute to the transfer signal. Excess dsDNA fragments and the released short ( +)strands comprise the fastest moving band detected in an autoradiogram after separation of the products by gel electrophoresis.

pletely saturate 40 ng of cssM13 DNA. This corresponds to one RecA monomer per 5 nucleotides. As can be seen in Fig. 2 , the lower limit of the assay (containing 40 ng cssDNA) is less than 300 ng RecA protein which yields a transfer signal of approximately 5 % of the total radioactivity. The sensitivity of the assay for the purified RecA protein would be considerably improved by reducing the amount of cssDNA. This would, however, not be the case when the assay is used to detect RecA activity in crude or partially purified nuclear extracts in which other DNA binding proteins may interfere with the adequate coating of the cssM13 DNA with RecA-like proteins.

Fig. 2. Protein titration curve of the strand-transfer reaction. Each reaction (30pI) contained 40ng of cssM13 DNA, 0.75-0.90 ng of 32P-labeled homologous dsDNA fragments, ATP ( 1 mM), an ATP regenerating system ( 18 mM creatinephosphate, 60 units/ml creatine kinase), and 12.5mM MgCI,. The reactions were started by addition of the indicated amounts of RecA protein, 1 pg RecA protein corresponding to one RecA monomer per 4 nucleotides. After 30min, the reactions were stopped and treated with proteinase K. The reaction products were separated on agarose gels and the radioactivity in products and substrate was quantified by transmission measurements using a Shimadzu, Dual wavelength TLC scanner. The amount of strand-transfer product is expressed as the relative amount of label at the position of cssM13 DNA.

Strand-transfer activity of histone H1 Calf thymus histone H I stimulates strand-transfer activity probably because of its ability to form large aggregates of both double- and singleand KOLODNER1990), stranded DNA (NORRIS and it is furthermore known to promote renaturation of complementary DNA strands (Cox and LEHMAN1981). Therefore, as a final control of the labeled DNA preparation the strand-transfer assay was performed under standard conditions in the presence of increasing amounts of HI protein, but in the absence of ATP and an ATP regenerating system. A distinct peak at the position of MI 3 ssDNA, indicating strand-transfer activity, was seen after addition of 10-50 ng HI protein, whereas higher concentrations of H1 inhibited the strand-transfer reaction, causing the aggregated labeled DNA to remain in the application slot of the gel (Fig. 3). This indicates that low concentrations of histone H1 are indeed able to catalyze strand-transfer of complementary DNA strands, but that addition of higher amounts of protein inhibits this activity, probably because of the H1-mediated formation of DNA aggregates.

Hereditas If 7 (1992)

STRAND-TRANSFER ACTIVITY IN BOMBYX MOM

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amount of extracted and partly purified proteins by 10 %. The amount of crude nuclear proteins extracted from 4th and 5th instar Bombyx testes (2700 pairs) was 0.4 % of the starting material. A second extraction in the presence of 80 mM DTT increased the yield to 0.5 % of the starting material. The two extracts were combined prior to hydroxylapatite chromatography. Detection of strand-transfer activity Strand-transfer activity was readily demonstrated both in crude 0.35 M NaCl extracts (not shown) and in partially purified extracts from Drosophila Fig. 3. Detection of strand transfer activity of calf thy- Kc cells. The activity of the crude extracts was mus histone HI. Each reaction (30 pl) contained 40 ng of approximately 75 % higher when the proteins were cssM 13 DNA, 0.35-0.45 ng of 32P-labeled homologous dsDNA fragments and 12.5 mM MgCl,. The amount of extracted in the presence of high concentration of protein was as follows. Lane I: no protein, lanes 2-6: 10, DTT. After hydroxylapatite chromatography, the 25, 50, 75, and 100 ng of histone HI. The assay condi- activity was, however, approximately the same for tions were as described in the legend to Fig. 2. both concentrations of DTT (Fig. 5, lanes 3 and 4), indicating that a stimulatory cofactor, released in the presence of 80mM DTT, is removed by Preparation of nuclear extracts hydroxylapatite chromatography. In 0.5-1.0 m M DTT protein extracts prepared Nuclear extracts were prepared and partially purified from Drosophilu tissue culture cells and from testes of Bombyx larvae at 3-4th, 4th, and 5th from whole Bombyx testes. No attempts were instar, strand-transfer activity is barely detectable made to fractionate the meiotic cells from Bombyx (Fig. 4, lanes 2-4), whereas extraction of nuclear testes. Ultrastructural analyses have shown that proteins in the presence of 80mM DTT yields a meiosis in male Bombyx commences during the 3rd distinct signal for strand-transfer at each of the larval instar by a series of synchronous mitotic three larval stages (Fig. 4, lanes 5-7). Assays for divisions of secondary spermatogonia, producing strand-transfer of the combined partially purified 64-cell clusters of primary spermatocytes. The nuclear extracts (Fig. 5) show a threefold increase number of somatic cells in 4th and 5th instar of activity when calculated per 10pg protein exlarvae is in this context negligible, the vast majority tract. Furthermore, it shows that even the lowest of cells being in various stages of meiotic prophase amount of protein (lane 5) appears to saturate the cssM13 DNA as two- or fourfold higher amounts (HOLMand RASMUSSEN1980). The preparation of nuclear extracts involved iso- of protein (lanes 6 and 7) fail to increase the lation of nuclei and extraction of nuclear proteins intensity of the signal for strand-transfer. Bombyx testes in early third instar larvae, i.e., by 0.35 M sodium chloride in the presence of either 0.5- 1.O or 80 mM DTT. The high concentration of prior to or at the onset of meiosis, are small and it DTT was chosen to promote the disintegration of was not possible to isolate sufficient material for the chromosomal scaffolds and, in case of meiotic further purification of the 0.35 M NaCl nuclear prophase nuclei, the synaptonemal complex (IER- extract. The activity of crude extracts (0.35 M NaCI/ 0.5 mM DTT)from larvae at this stage was 40 times ARDI et al. 1983). The crude protein extract was either assayed for strand-transfer activity directly higher than that of extracts prepared in the same way or further fractionated by hydroxylapatite chro- from 4th instar larvae (Fig. 4, lanes 1 and 3). This matography, which removes approximately 50 YO shows that a strand-transfer activity is readily extracted from Bombyx testes before the onset of of the soluble proteins. Thirty grams of Drosophilu tissue culture cells meiosis by 0.35 M NaCl in the presence of 0.5yielded about 40 mg protein after hydroxylapa- 1.0mM DTT but that the extraction of this activity tite chromatography, representing approximately from meiotic nuclei requires conditions in which the 0.1 % of the starting material. A second extraction chromosome scaffold and/or the constituents of the in the presence of 80mM DTT increased the synaptonemal complex are dissolved.

6 B. WISCHMANN

Fig. 4. Detection of strand-transfer activity in crude nuclear extracts of Bombyx testes. The amount and source of protein were as follows. Lane I: 4.3 pg from 3rd instar testes (containing mainly mitotic cells), lanes 2-4: 54,41, and 61 pg protein extracted with 1.0 mM DTT/0.35 M NaCl at 314th. 4th and 5th instar respectively, lanes 5-7: 13, 11.5, and 16 pg protein extracted with 80mM D l T / 0.35 M NaCl at the same stages as in lanes 2-4. The amount of substrate DNA and the assay conditions were as described in the legend to Fig. 2. The calculated amount of strand-transfer activity per 10 wg protein extract added is, lane I: 40.5 %, tunes 2-4: 0 %. 0.9 %, and 0 % and lanes 5 - 7 9.8 YO. 15.5 %, and 4.4 YO.

Immunological analysis The polyclonal anti-RecA antibody identified a single polypeptide in cell-free extracts of E. coli strain DR1453 with an apparent molecular weight of 38 kDa. corresponding to the size of the RecAprotein (HORIIet al. 1980). The specific antibodies were used in immunoblot assays to assess whether the crude or the partially purified nuclear extracts .~ proteins rrom Drosopphila and B o n i b ~ contained which cross react with the anti-RecA antibody. Up 10 200jig of protein from crude o r partially purified nuclear extracts was separated by SDSPAGE. transferred to nitrocellulose membranes, and probed with the antibody. The amount of protein loaded on the gel contained strand-transfer activity far in excess of the amount of RecA activi t y used to test the specificity of the antibody. None of the immunoblot assays revealed cross reaction between the anti-RecA antibody and the nuclear extracts and it is concluded that the RecAprotein and the strand-transfer activity of the Drosopliiltr and Bonih?,.~nuclear extracts are immunologically different.

Heredims 117 (1992)

Fig. 5. Detection of strand transfer activity in partially purified nuclear extracts from Drosophila Kc cells and Bombyx testes. The proteins were extracted with 80 mM

DlT/0.35 M NaCl and passed over a hydroxylapatite column as described in Materials and Methods. The amount and source of protein were as follows. Lane I : no protein, tune 2: 1.4pg RecA, lanes 3-4 4.4pg Drosophila protein extracted with 0.5 mM and 80 mM DTT, respectively, lanes 5-7: 1.7, 3.3, and 6.6pg of Bombyx protein from 4th and 5th instar testes, lane 8: 0.4 pg from the trailing edge of the peak eluted from the hydroxylapatite column. The amount of substrate DNA and the assay conditions were as described in the legend to Fig. 2.

Discussion In addition to its ATP-dependent strand-transfer activity, the E. coli RecA-protein promotes the renaturation of complementary D N A strands ( WEINSTOCKet al. 1979). Renaturation proceeds optimally in the presence of ATP a t a ratio of RecA-protein to ssDNA corresponding to 1015 % saturation. A smaller (approximately 50 7') RecA-promoted stimulation of renaturation does, however, occur also in the absence of ATP. One of the key points in evaluating the present results is therefore the reliability of the strand-transfer assay in distinguishing renaturation of single-stranded D N A and srrand-transfer. In the former case, the radioactive band at the position of cssM13 D N A would reflect a simple renaturation between possible contaminating radiolabeled ( -)strands in the dsDNA probe and the circular (+)strand of M13 DNA, whereas the strand-transfer reaction in-

Heredim I I7 (1992)

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volves the invasion of the single-stranded M 13 strand-transfer activity, whereas strandAtransfer acmolecule into a radiolabeled duplex molecule and tivity is readily demonstrated when extraction is the displacement of the homologous strand to pro- performed in the presence of 80 mM DTT. The alternative interpretation is considered unduce a plectonemic joint. The problem is emphasized by the recent report likely that this increase in recA-like activity upon of KAWASAKI et al. (1989), who demonstrated that extraction with 80 mM DTT in meiotic cells is the crude protein extracts prepared from calf thymus effect of changes in the relative amount of DNA as well as purified histone HI catalyze ATP-inde- binding proteins other than recA-like proteins. Mipendent strand-transfer. The conclusion was, how- totic nuclei of both Drosophilu and Bombyx (i.e., ever, recently retracted by the authors (KAWASAKI 3rd instar testes) release recA-like proteins upon et al. 1990) based on the finding of small amounts extraction in the presence of 0.5-1 mM DTT. Parof ssDNA contaminating the labeled dsDNA tial purification of the nuclear extracts from probe. When the assay for strand-transfer activity Drosophila by hydroxylapatite chromatography of HI was repeated with a ssDNA-free substrate, does not significantly change the specific activity of only trace amounts of strand-transfer were de- the extract. The presence of 80mM DTT in the tected. It was therefore concluded that histone H1 extraction buffer increases the specific activity by only mediates renuturution of complementary sin- 75 % compared to 800 % for the meiotic extract. gle-stranded DNA molecules. Although extraction with 80 mM DTT and partial As described by SVARENet al. (1987) ethanol purification by hydroxylapatite chromatography precipitation of short (93 bp) dsDNA fragments were not performed for 3rd instar Bombyx testes followed by drying in a lyophilizer or under vacuum (mitotic cells) it is considered likely that the result at room temperature led to the formation of aber- would be similar to those of mitotic Drosophilu rant ssDNA molecules, whereas ethanol precipita- cells. These observations thus indicate that neither tion without subsequent dehydration fails to extracting the nuclei with 80 mM DTT instead of produce the aberrant ssDNA molecules. The ds- 0.5- 1 mM DTT nor the fractionation over hydroxDNA substrate used in the present assay for strand- ylapatite influences the ratio of possible competing transfer was prepared, isolated, and radiolabeled DNA binding proteins and strand-transfer mediatfollowing procedures which are not expected to ing proteins to any major extent in extracts from promote denaturation and formation of single- mitotic cells. stranded hairpin structures. This was confirmed by In contrast to the crude Drosophilu extracts, electrophoresis on a non-denaturing 5 YOpolyacryl- 0.5- 1 mM DTT was insufficient to release proteins amide gel which, within the limits of resolution, from meiotic cells with a specific strand-transfer failed to reveal any trace of signal at the position of activity detectable with the assay. Fractionation of ssDNA fragments. The present study therefore 0.5-1 mM DTT meiotic extracts over hydroxylshows that histone HI indeed mediates a strand- apatite was not performed and it is not known whether this would increase the activity to an transfer of complementary DNA strands. With the aid of a sensitive assay for strand- extent making it detectable by the assay. Fractiontransfer activity in crude protein extracts, the pres- ation of the combined 0.5-1 and 80mM DTT ence of an ATP-independent activity in protein meiotic extracts, in contrast to extracts from extracts from mitotic Drosophilu nuclei (MC- mitotic cells, increases the specific activity about CARTHYet al. 1988) have been confirmed. A simi- eightfold. Thus, in meiotic cells the ratio of strandlar ATP independent activity was identified in transfer activity and competing DNA binding proteins extracted from 3rd instar Bombyx testes proteins and/or the amount of strand-transfer accontaining mainly mitotic cells. It is further shown tivity extractable by 80 mM DTT is significantly that conditions in which the chromosome scaffold different from that of mitotic cells. The difference in extraction conditions required dissolves (80 mM DTT) results in the release of additional proteins stimulating the strand-transfer to release/detect strand-transfer activity from soreaction. This effect was, however, not observed matic Drosophila and meiotic Bombyx nuclei as after fractionation of the extract over hydroxyl- well as the different response to purification on apatite. hydroxylapatite, indicate that different forms of Extraction of proteins from meiotic Bombyx nu- strand-transfer activity and/or competing DNA clei (4th and 5th instar larvae) with buffers con- binding proteins are present in the two cell types taining 0.5- 1.O mM DTT releases virtually no and/or that the nuclear localization of the activity

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differs, the meiotic form being associated with the synaptonemal complex or the chromosome scaffold in a way rendering it unextractable by 0.35 M NaCl unless the structural framework of the meiotic chromosomes is disrupted by high concentrations of DTT. The addition of increased amounts of meiotic Bombyx proteins does not lead to an increase of signal for strand-transfer, implying that even the smallest amount of protein used in the assay is sufficient for completely saturating the cssDNA. The maximum signal for strand-transfer in a standard assay is only about 10 % of that detected with the RecA protein under optimal conditions, which indicates that other ssDNA-binding proteins are present in the extract, preventing proper coating of the cssDNA with RecA-like proteins. The transfer activity extracted from meiotic Bornbyx nuclei does not require exogenously added ATP as is the case for the transfer activity isolated from meiotic and mitotic yeast cells (KOLODNER et al. 1987; SUGINOet al. 1988). whereas the activity isolated from meiotic cells of mouse and lily (HOTTA et al. 1985) has an absolute requirement for ATP. The strand-transfer activity identified in crude and partially purified extracts from Drosophilu embryos (MCCARTHYet al. 1988; EISEN and CAMERINI-OTERO 1988) and subsequently purified to near homogeneity (LOWENHAUPT et al. 1989) carries out the transfer reaction without a requirement for ATP. The uptake/assimilation of ssDNA by superhelical dsDNA catalyzed by the rec- 1 protein isolated from mitotic cells of Ustilago occurs in the absence of ATP (but is stimulated by ATP) whereas the reaction between linear dsDNA and circular ssDNA has an absolute requirement for ATP ( KMIECet al. 1985). Using linear dsDNA and homologous circular ssDNA as substrates in the strand-transfer assay, two activities have &en identified in human cells, one apparently ATP-independent ( H et al,~ 1986). ~ the ~other ~requiring ATP ( FISHEL et al. 1988). It is conceivable that these apparently conflicting .. results imply the existence of more than one protein catalyzing the strand-transfer reaction in mitotic as well as in meiotic cells. Several reports have described in detail the structural aspects of synapsis and synaptonemal complex formation at the early meiotic prophase and have documented in a variety of species that the resulting bivalent formation at pachytene is surprisingly regular (WETTSTEIN et al. 1984; JOHN

1990). In organisms where the early phases of synapsis have been studied, the homologous chromosomes are located randomly in the nucleus prior to synapsis (WETTSTEINet al. 1984) and extensive homology search is therefore required to achieve a proper synapsis. It is a common assumption that homology is checked by direct comparison of DNA sequences (SMITHIESand POWERS 1986; CARPENTER 1987; STERNand HOTTA1987). This assumption is corroborated by the presence of RecA-like proteins in nuclei at the meiotic prophase. Such proteins are capable of forming entities with ssDNA, which, through formation of paranemic joints with dsDNA molecules, can search dsDNA for regions of homology specified by the ssDNA. Once homologous sequences are aligned, the association between the homologous chromosome regions may be stabilized by formation of heteroduplex DNA. Whether or not simple gene conversion occurs by mismatch correction in such transient plectonemic joints and thus occur as “byproducts” of the recognition process as proposed by SMITHIES and POWERS (1986) and CARPENTER (1987) and reciprocal crossing over thus represents causally unrelated events, physically coupled to large/late recombination nodules, remains to be seen. A high strand transfer activity in the present study was obtained from testes of 4th instar larvae in which the majority of the cells are in the early meiotic prophase (HOLMand RASMUSSEN1980). The observation that extraction of protein( s) catalyzing strand-transfer from meiotic nuclei is facilitated when the chromosomal scaffold and the synaptonemal complex are dissolved, indicates that the strand-transfer activity is physically associated with these structures or their precursors. Acknowledgements. -- I would like to thank Prof. D. von Wettstein for suggesting this project and for his constant support and encouragement, Dr. C. G. Kannangara for help and advice with the purification of the RecA protein, Dr. S. W. Rasmussen for critically reviewing the manuscript, and B. S. Anderson and J. Sage for technical assistance. The work was supported by grant 816-168-DK from the Commission of the European Communities.

References BERGER,S. L. and KIMMEL, A. R. 1987. Guide to Molecular Cloning Techniques, Methods in Enzymology. - Arad. Press Inc., 152 p. CARPENTER, A. T . C. 1987. Gene conversion, recombination nodules and the initiation of meiotic synapsis. -- BioEssays 6 232-236 C o x , M. M . and LEHMAN,I. R. 1981. Renaturation of DNA: a novel reaction of histones. - Nucleic Acidr Res. 9 389-400

Hereditas 117 (1992)

EISEN,A. and CAMERINI-OTERO, R. D. 1988. A recombinase from Drosophila melanogaster embryos. - Proc. Natl. Acad. Sci. USA 85 7481-7485

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STRAND-TRANSFER ACTIVITY IN BOMBYX MORI

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RecA-like strand-transfer activity at the meiotic prophase in Bombyx mori.

An ATP-independent strand-transfer activity has been identified in nuclear extracts prepared from Drosophila tissue culture cells and isolated nuclei ...
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