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1 IUBMB Life, 48: , 1999 Copyright c 1999 IUBMB /99 $ Original Research Article 5 0 to 3 0 Single Strand DNA Exonuclease Activity in a Preparation of Human Ku Protein Viktor E. Morozov and Brian G. Fuller Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Building 10, Room B3-B69, Bethesda, Maryland 20892, U.S.A. Summary We describe a novel 5 0 to 3 0 single-strand exonuclease activity exhibited by a Ku preparation puri ed from a human cell line. The enzyme removes 5 0 single-strand extensions from duplex DNA molecules. The exonuclease and helicase activities respond reciprocally to changes in ATP concentrations: Nuclease activity is inhibited at the ATP concentrations that are optimal for the helicase. The exonuclease activity does not require divalent cations. The potential implications of the exonuclease activity ndings for repair of double-strand breaks and recombination processes are discussed. IUBMB Life, 48: , 1999 Keywords DNA-PK; double-strand DNA break; exonuclease activity; Ku protein. INTRODUCTION Mammalian Ku is a DNA-binding protein (1) essential for repair of double-strand break (DSB) 1 and V(D)J recombination (2, 3). The protein is a heterodimer of 70- and 86-kDa subunits (1) that, in the presence of double-strand (ds) DNA, combines with a 460-kDa catalytic subunit, DNA-PK CS, to form the DNAdependent serine/threonine protein kinase DNA-PK (1, 4). Ku serves the dual function of DNA-binding and activation of the catalytic subunit (4). Mutational analysis in rodent cells has de- ned X-ray complementation groups (XRCCs), the products of which are essential for both general DSB repair and V(D)J recombination. Ku p86 and DNA-PK CS have been identi ed with XRCC5 (5) and XRCC7 (6), respectively, and Ku p70 corresponds to XRCC6 (7). Mutations in DNA-PK CS are responsible Received 4 October 1999; accepted 7 October Address correspondence to Viktor Morozov. Fax: (301) viktorm@box-v.nih.gov 1 Abbreviations: DNA-PK, DNA protein kinase; DNA-PK CS, DNA-PK catalytic subunit; ds, double-strand; DSB, double-strand break; SDS, sodium dodecyl sulfate; ss, single-strand; XRCC, X-ray complementation groups. for the defective V(D)J recombination and DSB repair in scid mice (4, 6). Certain properties of Ku seem relevant to its possible role or roles in DNA repair. Ku binds tightly to the ends of dsdna (1, 8, 9) and to internal points of single-strand to double-strand (ss/ds) transition such as nicks and larger ss gaps, bubbles of nonhomology, and hairpin con gurations (9, 10). Ku bound at the ends of dsdna can translocate inward in an ATP-independent fashion. However, if the catalytic subunit is also present, the DNA-PK complex can translocate inward only in the presence of ATP (11, 12). Both endogenous and recombinant Ku preparations have been shown to exhibit an ATP-Mg 2+ -dependent helicase activity in vitro (13). A ssdna dependent ATPase activity associated with Ku puri ed from HeLa cells (14) has subsequently been identi ed as the helicase (15). Several Ku mutations that do not affect the known properties of Ku fail to complement repair de ciency in vivo (16, 17), suggesting that Ku has additional, yet to be identi ed biochemical activities that are important for DSB repair. Several models have been proposed for rejoining of DSBs (18 24); all implicate the participation of 3 0 ss tails. In DSB repair by homologous recombination in yeast, a 5 0 to 3 0 exonuclease activity is postulated (18, 20), followed by 3 0 ss invasion at the homologous regions of the undamaged chromosomal DNA, generation of Holliday junctions, and gene conversion (25). Mammalian cells preferentially repair DSBs by nonhomologous recombination (26), in which end-joining of 3 0 ss tails is mediated by microhomology of 1 to 6 bases at the recombinant junction (27). Here again, a 5 0 to 3 0 exonuclease activity is thought to carry out strand recission to generate the 3 0 OH unmatched strands. We have found 5 0 to 3 0 exonuclease activity in a preparation of human Ku protein. MATERIALS AND METHODS Protein Preparation. Ku protein was prepared from the human 293 cell line nuclear extracts. Puri cation procedure and 593

2 594 MOROZOV AND FULLER chromatographic steps: heparin Sepharose (Amersham Pharmacia Biotech), MonoQ (Amersham Pharmacia Biotech), and two sequence-speci c DNA-af nity columns were described in details early (9, 28). Recombinant protein was puri ed from Drosophila Sf9 cells cotransfected with Baculovirus constructs encoding the human p86 and p70 Ku subunits (29). Nucleic Acid Constructs. The 3 0 ends were labeled by addition of 32 P-cordycepin with use of terminal transferase (Promega) according to the directions of the manufacturer, and the 5 0 ends were labeled with c 32 P-ATP and T4 polynucleotide kinase. For labeling with a 32 P-cordycepin, ssdna was phosphorylated with nonradioactive ATP and T4 polynucleotide kinase before labeling. The 100-nt fragment was derived from nucleotides 1051 to 1150 of M13 viral DNA, with the following substitutions: at , CGGT becomes GCCA; at 1120, G becomes A; at 1122, G becomes T; at , AGGCG becomes TATAT. The ss portions of the smaller oligonucleotides were anti-complementary to the 100-nt component in those positions. Labeled strands were annealed to the unlabeled partners and the products were isolated by electrophoresis in 15% polyacrylamide nondenaturing gels. Nuclease Assay. The assay conditions were those of Tuteja et al. (13) for measurement of Ku-associated helicase activity: The reaction mixture (7.5 l l) contained 20 mm Tris-HCl, ph 8.0, 1 mm MgCl 2, 60 mm KCl, 4 mm or 0 mm ATP, 2 mm dithiothreitol, 0.2 mm EDTA, 5% glycerol, 0.15 ng (unless otherwise mentioned) of DNA construct (Fig. 1) and (unless otherwise speci ed) 20 ng of Ku preparations puri ed from human 293 cells (9, 28) or human recombinant Ku p70 and Ku p86 kda Ku subunits (29). The enzyme reactions were incubated at 37 ± C for 30 min, unless otherwise indicated, and the products of the reactions were fractionated on a nondenaturing 4 20% polyacrylamide gradient or on 20% polyacrylamide sequencing gels and then autoradiographed. In the antibody inhibition experiments, 0.8 l g of individual commercially available puri ed mouse monoclonal antibodies (Lab Vision Corp.) was used in a standard nuclease reaction mixture containing Ku preparation from human 293 cells. Antibody was preincubated with Ku in standard nuclease reaction mixture for 5 min at 37 ± C before construct a 0 was added. The products of the nuclease reaction, which represented the nuclease activity, were separated on a 20% polyacrylamide sequencing gel. The autoradiographic lm densities of the reaction products were analyzed with the NIH Image Program Version In the experiments evaluating substrate requirements and ATP dependence, Ku protein was added at 133 fmol per reaction. The mixtures were incubated for 30 min, and intact substrate and the reaction products were separated on a 20% polyacrylamide sequencing gel before autoradiography. The lm densities in the positions of the uncut substrates and products of reaction were measured by using a video camera and the NIH Image Program. The loss of autoradiographic lm density of the uncut substrates and the increased lm density of the products of the reaction were considered to represent the nuclease activity. Figure 1. Constructs used as nuclease substrates. The position of the 32 P label is indicated by a lled circle. Gel-Filtration Separation and Gel Mobility Shift Assay. Ku protein from human 293 cells was examined by gel ltration on a commercially prepacked HiLoad 16/60 Superdex 200 prepgrade column (Amersham Pharmacia Biotech). Separation was performed in the column buffer (28), ow rate was 0.5 ml/min, and the fraction size was 1 ml. Aliquots of 4 l l from each fraction of the Ku protein peak, which eluted between fractions 44 and 50 (44 50 ml), were analyzed in a gel mobility shift assay for DNA-binding activity (performed as described in ref. 9) and were assayed for the presence of exonuclease activity as described above with construct a 0 used as substrate. RESULTS AND DISCUSSION The substrate constructs shown in Fig. 1 resemble those used by Tuteja et al. (13) in demonstrating Ku-associated helicase activity. However, we used a synthetic 100-nt ssdna as the larger partner in the constructs (see Materials and Methods) rather than the whole M13 viral DNA (13). In this study we used Ku prepared from the human 293 cell line (9, 28) and recombinant Ku protein puri ed from Drosophila Sf9 cells cotransfected with Baculovirus constructs encoding the human p86 and p70 Ku subunits (29). Silver-stained patterns of both Ku preparations after electrophoresis on the sodium dodecyl sulfate (SDS) containing gradient polyacrylamide gel are shown in Fig. 2.

3 5 0 TO 3 0 SSDNA EXONUCLEASE ACTIVITY IN KU PROTEIN 595 Figure 2. Silver-stained 4 20% gradient SDS polyacrylamide gel analysis of the Ku proteins. Lane 2, Ku recovered from Drosophila Sf9 cells cotransfected with Baculovirus constructs encoding the human 70- and 86-kDa Ku subunits (29); lane 3, Ku p70 and p86 with contamination by 460-kDa DNA- PK CS puri ed from human 293 cells (9, 28); lanes 1 and 4, molecular mass markers. To assess the helicase activity of our Ku preparation from 293 cells, we incubated construct a (Fig. 1) with Ku under the reaction conditions described (13; see Materials and Methods) for helicase activity (4 mm ATP, 1 mm MgCl 2 ) and the products were monitored on a nondenaturing polyacrylamide gel (Fig. 3). Displacement of the labeled 47-nt fragment ( helicase product ) was observed. A small amount of more rapidly moving material was also present. When ATP was omitted, helicase activity was abolished, and the smaller electrophoretic component ( nuclease product ) was predominant. Construct a incubated with Ku protein from the human 293 cells resulted in a mobility shift that was consistent with nuclease cleavage. This effect was greater when ATP was omitted, which re ects degradation of the DNA construct without strand separation by helicase activity. Recombinant Ku showed no increase in 47-nt fragment ( helicase product ) in the presence of ATP but produced a distinctly smaller component in the absence of ATP. Recombinant Ku puri ed from Sf9 cells displayed less nuclease activity, but there was no difference in reaction product mobility for both preparations of Ku protein. An impaired helicase activity was previously reported (13) for another recombinant Ku preparation of this type (29). We performed several experiments to try to understand the nature of this activity. To identify the active polypeptide, we stud- Figure 3. Reciprocal helicase and nuclease activities, depending on the presence or absence of ATP. The reaction mixture, reproducing the conditions used for the Ku helicase reaction (13), contained ATP where indicated, 0.1 ng of construct a (Fig. 1), and 40 ng of Ku preparation puri ed from human 293 cells (9, 28) or 20 ng of human recombinant p70 and p86 Ku subunits (29). The products of the reactions were fractionated on a nondenaturing 4 20% polyacrylamide gradient gel. ied suppression of nuclease activity with commercially available well-established puri ed mouse monoclonal anti-ku antibodies and with construct a 0 as the substrate (Fig. 4A). The reaction products (labeled mononucleotides) were analyzed on a sequencing gel. As a negative control, similarly puri ed anti-p53 antibody obtained from the same supplier was used at the same concentration. The presence of control IgG to p53 resulted in a 20% inhibition of the nuclease reaction. This was comparable with the 20% inhibition caused by anti-ku 70 kda (which recognizes the epitope sequence between amino acid residues 506 and 541 of Ku 70 polypeptide). Approximately 45% inhibition was observed with an antibody that recognized the conformational epitope of the Ku 70/86 dimer. Antibody to C-terminal region of Ku 86-kDa polypeptide, which binds an epitope between amino acid residues 610 and 705, displayed more than 71% inhibition and was the most effective among the tested IgG (Fig. 4A), being 50% more effective than background suppression with nonspeci c antibodies. The partial inhibition of activity suggests that amino acid residues between 610 and 705 of the Ku 86-kDa polypeptide are not a part of the active center of the nuclease but perhaps reside close enough to the active center to limit access of substrate by 50% when this epitope is blocked by antibodies. Substrate requirements for the rst complete reaction cycle were examined by titrating 133 fmol of Ku protein (17 nm) with increasing amounts of construct a 0, labeled at the 5 0 end of the short strand (Fig. 1). In this case, the labeled reaction products on the sequencing gel were the nucleotide monophosphate and a barely detectable amount (if any) of the dinucleotide. Activity (release of labeled mononucleotides from the 47-bp strand in 30 min) plateaued at 150 fmol of a 0, i.e., at an approximately equimolar ratio with Ku (Fig. 4B). Ku binds in

4 596 MOROZOV AND FULLER (A) (B) (C) Figure 4. (A) Inhibition of Ku exonuclease activity by monoclonal anti-ku antibodies. Exonuclease reaction products (mononucleotides, referring to markers in the right lane), which represented the nuclease activity, were separated on a 20% polyacrylamide sequencing gel. (B) Substrate requirement of the Ku nuclease reaction. A constant amount of labeled construct a 0 was diluted with increasing amounts of nonradioactive construct and added to the ATP-minus reaction mixtures in the amounts shown. The autoradiographic lm densities of the mononucleotide-reaction products were plotted in arbitrary units on the ordinate. (C) Characterization of Ku protein in gel- ltration separation fractions from the HiLoad 16/60 Superdex 200 column.

5 5 0 TO 3 0 SSDNA EXONUCLEASE ACTIVITY IN KU PROTEIN 597 a sequence-independent manner to oligonucleotide constructs similar to a 0 with dissociation constants in the pm range (8, 9) and remains bound to the substrate after completing the initial cleavage step. Nuclease activity follows Ku through the high-salt, multiple ion-exchange and af nity isolation steps of the protein preparation from nuclear extract (9, 28). The classical way to quantitatively monitor a 5 0 to 3 0 Ku-associated nuclease activity throughout the puri cation scheme is limited by the inability to distinguish between Ku-associated and other, similar nuclease activities in the crude nuclear extract and in the earliest steps of puri cation. Instead, therefore, we studied the nature of the nuclease activity by fractionating the Ku protein from human 293 cells on a gel- ltration column, a method that had not been used as a puri cation step in the Ku preparation (9, 28). The 5 0 to 3 0 nuclease activity eluted in fractions from the prepacked HiLoad 16/60 Superdex 200 column matched the elution pro le of DNA-binding activity of Ku protein (Fig. 4C). The nuclease reaction product formed from construct a in the absence of ATP (Fig. 3) was resolved on a sequencing gel (Fig. 5A) into a short ladder of discrete components with nucleotide lengths of 32 and 33 nt (major components) and 34 to 36 nt (minor). These fragments represent cleavage of the 3 0 -labeled 47-nt fragment in a at its 5 0 ss/ds junction or 1 to 4 nt upstream from there. Phosphorylation of the 5 0 hydroxyl group was required for nuclease activity (data not shown). The nature of the cleavage reaction was further investigated with constructs b, c, and d (Fig. 5A). The completely basepaired ds region of 32-nt component of construct b was not cleaved, con rming the 5 0 ss requirement for nuclease activity. In constructs c and d, the 100-nt component was labeled at its 3 0 end. Digestion with Ku gave labeled products with apparent sizes of 67 and 82 nt, respectively, consistent with cleavage of the 5 0 ss portion of the 100-nt component in each construct. In experiments not illustrated here, we found that a blunt-ended 32-bp duplex was not modi ed under our reaction conditions. Similarly resistant to digestion were a 100-bp ds fragment containing a 12-nt internal bubble of nonhomology and a 33-bp blunt-ended duplex hairpin with a 4 nt ss loop both constructs to which Ku is also known to bind tightly (9). The time course of the nuclease reaction is shown in Fig. 5B. In this experiment the labeled component of construct a was a mixture of 47- and 46-nt strands. Most of the labeled strands were cleaved in the rst 5 10 min of the reaction, yielding fully trimmed products of 31 and 32 nt as well as a ladder of larger fragments ranging upward in size towards the remaining (A) Figure 5. (A) Nuclease activity towards substrates a to d in Fig. 1. Reaction conditions were the same as in Materials and Methods except that ATP was omitted and 80 ng of Ku protein was added. The reaction products were denatured, and the labeled strands were analyzed on a 20% polyacrylamide sequencing gel. Speci c activity of incorporated 32 P was less for constructs c and d than for constructs a and b. The sizes of labeled markers are indicated at the left. (B) Time course of the Ku nuclease activity toward substrate a in Fig. 1. Reaction conditions were as in Materials and Methods but with ATP omitted. Aliquots were taken at the indicated times for analysis on 20% polyacrylamide sequencing gel. (B)

6 598 MOROZOV AND FULLER Table 1 Ku exonuclease activity a with different nucleotide triphosphates b Percent of maximum activity No NTP ATP ATPc S GTP CTP UTP Construct a Small single strand of construct a c a Exonuclease activity is expressed as percent of substrate cleaved in the absence of NTP. b All nucleotide triphosphates were tested at a nal concentration of 5 mm. c Reaction conditions were the same as with dsdna constructs. Figure 6. ATP dependence of the Ku-associated 5 0 -exonuclease activity. Construct a (Fig. 1), 0.15 ng, was incubated with 20 ng of Ku at the indicated ATP concentrations and analyzed as described in Materials and Methods. The loss in lm density in the positions of uncut substrates (5 0 -exonuclease activity) was determined at each ATP concentration and plotted as the percent of the completed reaction. substrate. These heterogeneous fragments were further processed until the ladder of products had achieved its nal size distribution (by 30 min). This shows that the short ladder of fragment sizes is the nal product of the nuclease reaction. ATP was increasingly inhibitory to the nuclease activity at concentrations >0.1 mm (Fig. 6); at 4 5 mm, as is used in the helicase reaction (13), ATP was almost completely inhibitory (compare Figs. 3 and 6). Table 1 compares the inhibitory effects of other nucleotide triphosphates with that of ATP. All four of the natural triphosphates were strongly inhibitory at the concentration tested (5 mm). c -ThioATP was also a potent inhibitor, suggesting that the inhibition by ATP could result from an allosteric effect on the enzyme protein or from competition for the exonuclease catalytic site. In experiments not detailed here, we have found that ssdna is also a substrate for the nuclease (V. E. Morozov, unpublished data). This activity was also strongly inhibited by ATP, GTP, and thioatp but was somewhat more resistant to the pyrimidine triphosphates (Table 1). A similar pattern and extent of inhibition when ATP was replaced with CTP, GTP, or UTP was observed for Ku-associated helicase activity (13). Mg 2+ or Mn 2+ was required for helicase activity (13); however, omission of these cations or addition of 6 mm EDTA to the reaction mixture did not affect the nuclease activity. The exonuclease described here requires an overhanging 5 0 ss end and is inactive against blunt-ended DNA or DNA with a 3 0 overhang. In the case of DSBs, unwinding of DNA by the Ku- associated helicase activity could provide a correct substrate because this activity proceeds in the 3 0 to 5 0 direction with respect to the opposite strand (13). Both helicase and exonuclease activities may be simultaneously expressed at certain ATP concentrations (e.g., 0.5 to 2 mm) within the physiological range. Recombination intermediates with extended 3 0 ends were recovered after injection of linear duplex DNA with homologous ends into Xenopus leavis oocytes (22), suggesting the presence of a 5 0 to 3 0 nuclease activity that carries out strand recission as an early step in repair of DNA DSBs. It may be noted that Ku homologous proteins (88 kda and 72 kda) are presented in Xenopus eggs and cross-react with antibodies against the corresponding human subunits (30, 31). One must also consider whether the nuclease activity is an intrinsic property of Ku or is provided by a cofractionating (possible tightly bound) protein that followed Ku through the high-salt, multiple af nity and ion-exchange isolation steps involved in the preparation (9, 28). Five experiments described here provide supportive evidence that nuclease activity is a property of the Ku protein: (i) The Ku puri ed from the human 293 cells was devoid of DNA-PK activity (32), although the catalytic subunit (DNA-PK CS ) was observed on silver-stained polyacrylamide gels. No other components were visualized with molecular mass range as low as 5 7 kda and lower (Fig. 2). (ii) Preparation of recombinant Ku exhibits a 5 0 to 3 0 nuclease activity (Fig. 3). (i ii) Exonuclease activity coeluted with other wellestablished DNA-binding activity of Ku protein on gel- ltration column separation (Fig. 4C). (iv) Nuclease activity was partially suppressed by monoclonal antibodies against the Ku heterodimer and to greater degree by antibodies to the C-terminal epitope on the Ku 86-kDa subunit (Fig. 4A). (v) Ku is known to bind tightly to DNA molecules with either blunt or overhanging ends (8, 9) and reportedly remains associated with its processed substrate DNA during the course of the helicase reaction (13). The nding that maximal nuclease activity was reached when substrate and Ku were equimolar (Fig. 4B) is consistent with these properties of Ku protein. De nitive identi cation of the active polypeptide, as well as evidence that the exonuclease has a

7 5 0 TO 3 0 SSDNA EXONUCLEASE ACTIVITY IN KU PROTEIN 599 physiological role in vivo, requires further genetic and biochemical analysis. The results of exonuclease activity inhibition with anti-ku monoclonal antibodies suggest that the 86-kDa but not the 70-kDa polypeptide is associated with exonuclease activity, with localization of active center on or close to the C terminal ends. The 70-kDa subunit of Ku is primarily responsible for DNA binding (1). Because DNA-PK CS was present in the exonuclease active preparation of Ku protein from human 293 cells (Fig. 2), we cannot exclude the possibility that DNA-PK CS contributes to the exonuclease activity. In this communication we have not considered the possible role of DNA-PK CS, which is known to be essential for repair of DSB and is associated with Ku at DSBs. This protein kinase may regulate the activities of Ku, including Ku-associated helicase and exonuclease activities, through appropriately timed phosphorylations a possibility currently under study. All three DNA-PK subunit proteins are known to become phosphorylated when the holoenzyme is activated (4). ACKNOWLEDGMENTS The major part of this work was done in Laboratory of Biochemistry, NCI, NIH. We express our appreciation and thanks to Dr. Edward L. Kuff, Laboratory of Biochemistry, NCI, NIH, for help, and support of this work. We thank Drs. C. Norman Coleman, James B. Mitchell, and Angelo Russo, ROB, NCI, NIH for critically reading the manuscript. The Baculovirus constructs encoding the human p86 and p70 Ku subunits and some of recombinant Ku protein were kindly provided from Dr. Martin Gellert s Laboratory, NIDDK, NIH. REFERENCES 1. Reeves, W. H., Satoh, M., Wang, J., Chou, C. H., and Ajmani, A. K. (1994) Rheum. Dis. Clin. North Am. 20, Roth, D. B., Lindahl, T., and Gellert, M. (1995) Curr. Biol. 5, Weaver, D. T. (1995) Adv. Immunol. 58, Anderson, C. W., and Carter, T. H. (1996) Curr. Top. Microbiol. Immunol. 217, Taccioli, G. E., Gottlieb, T. M., Blunt, T., Priestley, A., Demengeot, J., Mizuta, R., Lehmann, A. R., Alt, F. W., Jackson, S. P., and Jeggo, P. A. (1994). Science 265, Sipley, J. D., Menninger, J. C., Hartley, K. O., Ward, D. C., Jackson, S. P., and Anderson, C. W. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, Gu, Y., Jin, S., Gao, Y., Weaver, D. T., and Alt, F. W. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, Blier, P. R., Grif th, A. J., Craft, J., and Hardin, J. A. (1993) J. Biol. Chem. 268, Falzon, M., Fewell, J. W., and Kuff, E. L. (1993) J. Biol. Chem. 268, Paillard, S., and Strauss, F. (1991) Nucleic Acid Res. 19, Calsou, P., Frit, P., Humbert, O., Muller, C., Chen, D. J., and Salles B. (1999) J. Biol. Chem. 274, de Vries, E., Van Driel, W., Bersma, W. G., Arnberg, A. C., and Van der Vliet, P. C. (1989) J. Mol. Biol. 208, Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N. W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., et al. (1994) EMBO J. 13, Cao, Q. P., Pitt, S., Leszyk, J., and Baril, E. F. (1994) Biochemistry 33, Vishwanatha, J. K., Tauer, T. J., and Rhode, S. L. (1995) Mol. Cell. Biochem. 146, Jin, S., and Weaver, D. T. (1997) EMBO J. 16, Singleton, B. K., Priestley, A., Steingrimsdottir, H., Gell, D., Blunt, T., Jackson, S. P., Lehmann, A. R., and Jeggo, P. A. (1997) Mol. Cell. Biol. 17, Ivanov, E. L., and Haber, J. E. (1995) Mol. Cell. Biol. 15, Keeney, S., Giroux, C. N., and Kleckner, N. (1997) Cell 88, Kramer, K. M., Brock, J. A., Bloom, K., Moore, J. K., and Haber, J. E. (1994) Mol. Cell. Biol. 14, Maryon, E., and Carroll, D. (1991) Mol. Cell. Biol. 11, Maryon, E., and Carroll, D. (1991) Mol. Cell. Biol. 11, Mason, R. M., Thacker, J., and Fairman, M. P. (1996) Nucleic Acids Res. 24, Thode, S., Schafer, A., Pfeiffer, P., and Vielmetter, W. (1990) Cell 60, Priebe, S. D., Westmoreland, J., Nilsson-Tillgren, T., and Resnick, M. A. (1994) Mol. Cell. Biol. 14, Sargent, R. G., Brenneman, M. A., and Wilson, J. H. (1997) Mol. Cell. Biol. 17, Roth, D. B., and Wilson, J. H. (1986) Mol. Cell. Biol. 6, Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, Ono, M., Tucker, P. W., and Capra, J. D. (1994) Nucleic Acids Res. 22, Higashiura, M., Takasuga, Y., Yamashita, J., and Yagura, T. (1993) Chromosome Res. 1, Someya, A., Take, Y., and Shioda, M. (1995) Biochem. Biophys. Res. Commun. 213, Morozov, V. E., Falzon, M., Anderson, C. W., and Kuff, E. L. (1994) J. Biol. Chem. 269,