Mutational Analysis of the Tfiiib-dna Target of Ty3 Retroelement Integration

The Ty3 retroviruslike element inserts preferentially at the transcription initiation sites of genes transcribed by RNA polymerase III (pol III). The requirements for TFIIIC and TFIIIB in Ty3 integration into the two initiation sites of the U6 gene carried on pU6LboxB were previously examined. Ty3 integrates at low, but detectable, frequencies in the presence of TFIIIB subunits Brf1 and TBP. Integration increases in the presence of the third subunit, Bdp1. TFIIIC is not essential but the presence of TFIIIC specifies an orientation of TFIIIB for transcriptional initiation and directs integration to the U6 gene-proximal initiation site. In the current study, recombinant wild-type TBP, wild-type and mutant Brf1, and Bdp1 proteins and highlypurified TFIIIC were used to investigate the roles of specific protein domains in Ty3 integration. The amino-terminal half of Brf1, which contains a TFIIB-like repeat contributed more strongly than the carboxyl-terminal half of Brf1 to Ty3 targeting. Each half of Bdp1 split at amino acids 352 enhanced integration. In the presence of TFIIIB and TFIIIC, the pattern of integration extended downstream by several base pairs compared to the pattern observed in vitro in the absence of TFIIIC and in vivo, suggesting that TFIIIC may not be present on genes targeted by Ty3 in vivo. Mutations in Bdp1 that affect its interaction with TFIIIC resulted in TFIIIC-independent patterns of Ty3 integration. Brf1 zinc ribbon and Bdp1 internal deletion mutants that are competent for pol III recruitment, but defective in promoter opening, were competent for Ty3 integration irrespective of the state of DNA supercoiling. These by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 4 results extend the similarities between the TFIIIB domains required for transcription and Ty3 integration and also reveal requirements that are specific to transcription. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 5 INTRODUCTION Ty3 is a gypsy-like retroelement in Saccharomyces cerevisiae (1). Despite similarities between the proteins encoded by Ty3 and other gypsy-like elements and retroviruses, Ty3 has the unusual property of inserting within a few nucleotides (nt) of the transcription start site of genes transcribed by pol III, including tRNA, U6, and 5S RNA genes. Mutations in the box A and box B promoter elements of tRNA and U6 RNA genes that interfere with transcription also diminish transposition in vivo, suggesting that active targets in vivo must be able to bind pol III transcription factors (2). Formation of the pol III transcription initiation complex (reviewed in 3-5) and Ty3 integration occur in close proximity to one another on DNA (2). The box A and box B promoter elements of tRNA and SNR6 genes serve to bind TFIIIC through sequence-specific interactions with two of the six TFIIIC subunits. The TFIIIC complex acts in turn to load the transcription initiation factor TFIIIB (6-8). TFIIIB is comprised of three subunits: the TFIIB-related factor Brf1, TBP and Bdp1 [previously referred to as B” and now designated Bdp1 for consistency with gene nomenclature (9)]. In vitro, as described in more detail below, TFIIIB can bind to SNR6 independently of TFIIIC (10,11). The positions of TFIIIC and TFIIIB subunits in promoter complexes have been mapped downstream and upstream, respectively, of the initiation site by crosslinking analysis (12,13). Ty3 strand transfer occurs at a site that is located between the positions occupied by the TFIIIC 120 kDa subunit (Tfc4) by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 6 on the downstream side and by the TFIIIB Bdp1 and Brf1 subunits on the upstream side. The strand transfer of the Ty3 3' end to the transcribed strand is typically between bp +1 and –1, while the strand transfer on the nontranscribed strand is between bp –5 and –6 (14,15), within the DNA segment that is strand-separated in the pol III open promoter complex (16). The in vitro requirements for Ty3 integration into tRNA genes have been probed using TFIIIC and TFIIIB. In the in vitro integration reaction, VLPs formed in yeast cells overexpressing Ty3 act as the source of integrase and full-length, extrachromosomal Ty3 DNA (17). The level of Ty3 integration into a plasmid-borne target in the presence of various test proteins is monitored by PCR. Transposition into a tRNA gene type target was shown to require TFIIIC and TFIIIB. Integration was negatively affected by pol III, indicating that Ty3 might resemble pol III in its requirements for target access, but that transcription initiation per se is not required (18). The promoter structure of the U6 RNA gene, SNR6, differs from that of most tRNA genes in that it contains an upstream TATA element (7,19). Although SNR6 expression in vivo requires TFIIIC, in vitro the strong SNR6 TATA box can directly mediate binding of TFIIIB through its TBP subunit. SNR6 can then be transcribed by pol III, independently of TFIIIC (11,20). In the latter context, TFIIIB binds to the nearly symmetric SNR6 TATA element in either orientation, and this can be monitored by transcription of a plasmid construct containing divergent transcription units (21,22). When TFIIIB and TFIIIC are present together, TFIIIC orients TFIIIB so that initiation by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 7 occurs predominantly at the SNR6-proximal site. Using a modification of the in vitro integration assay described above, it was shown that, similar to what is observed for transcription initiation, TFIIIB is sufficient to support Ty3 integration at the SNR6 transcription initiation site. In the presence of TFIIIC, Ty3 integration is specified by the predominant TFIIIB orientation (23). The ability to assemble TFIIIB sequentially on the SNR6 gene, together with knowledge of the TBP-DNA crystal structure and the availability of recombinant wildtype and mutant proteins has made it possible to delineate the roles of specific TFIIIB domains in pol III transcription initiation. TBP binds through sequence-specific interactions, sharply kinking DNA at both ends of its binding site (24,25). Aminoand carboxyl-terminal halves of Brf1 interact with the carboxyland amino-terminal lobes of TBP, respectively, and contact the DNA on either side of the TBP binding site to form the B' complex (26-29). Bdp1 binds primarily through contacts with the carboxyl-terminal half of Brf1, stabilizes the complex and probably brings DNA segments flanking the TATA element into closer proximity of one another. In the case of templates bound by TFIIIC, evidence suggests that both Brf1 and Bdp1 interact with TFIIIC (3,13,30,31). Brf1 and Bdp1 each contact pol III, although the primary specific contacts appear to occur through Brf1 (32-35). The apparently secondary role of Bdp1 is underscored by the observation that minimal transcription complexes supporting pol III initiation can be formed from TBP and Brf1 alone on DNA that is pre-melted at the initiation site (36). These results support the model that Bdp1 plays a primarily post-recruitment role in formation of the open transcription complex. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 8 Indeed, pol III transcription initiation complexes formed with certain Bdp1 mutants are defective at the promoter opening step (4,37). Using the in vitro integration system, it was previously shown that detectable SNR6 transposition targeting occurs with B' alone, but that the level of transposition is significantly increased by the addition of Bdp1 (23). Whether Bdp1 plays a significant role by stabilizing the transcription complex for targeting or by producing a local DNA structure that is conducive to integration was not determined. Insights concerning the roles of specific TFIIIB subunits and domains in pol III transcription provide a useful backdrop for designing experiments to probe the mechanism of the Ty3 integration reaction and explore the extent to which it resembles pol III recruitment and transcription initiation. The current study was undertaken using SNR6, highly purified TFIIIC, and recombinant wild-type and mutant TFIIIB subunits to address the following questions: Are the same domains in Brf1 and Bdp1 required for integration and transcription? Is the Bdp1 post-recruitment function in promoter opening required for Ty3 integration? How do interactions between TFIIIB and TFIIIC affect the pattern of Ty3 integration sites in vitro? The results of these studies extend the similarities in protein-DNA complex requirements between Ty3 integration and pol III transcription initiation, but identify interesting distinctions as well. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 9 MATERIALS AND METHODS Strains and Growth Conditions--Standard methods were used for culturing and transforming E. coli and S. cerevisiae (38). All plasmids were amplified in, and prepared from, E. coli HB101 [F ̄ hsdS20 (rB mB ) recA13 leuB6 ara-14 proA2 lacY1 galK2 rpsL20 (sm) xyl-5 mtl-1 supE44λ]. Ty3 was expressed in S. cerevisiae, NOY384, a gift from M. Nomura (University of California, Irvine), and transformed with the high-copy galactose-inducible plasmid, pEGTy3-1 (39). Plasmid Constructions--Recombinant DNA constructions and methods followed standard procedures (38). Plasmid pEGTy3-1 was used for galactoseinducible expression of Ty3 (39). Plasmid pLY1855 (23) was the target for Ty3 integration in vitro. Plasmids pDLC370 (2) and pLY1842 (23) served as PCR controls for integration into r-U6 and l-U6, respectively. Plasmid pDLC370 contains a Ty3 insertion upstream of SNR6 at r-U6 and plasmid pLY1842 is a clone containing an amplified fragment templated from a Ty3 insertion at l-U6. Supercoiled target DNA was prepared by centrifugation twice over cesium chloride density gradients, followed by chromatography over Sepharose CL2B. DNA was extracted with isopropanol saturated with cesium chloride followed by precipitation using standard methods. Linear integration targets were prepared by digesting pLY1855 (purified as described above) with the restriction endonuclease, HindIII. Digested DNA was extracted with phenol/chloroform and precipitated. DNA was resuspended in 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA. Linear DNA was checked for complete digestion by agarose gel electrophoresis. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 10 Protein preparations--Ty3 VLPs were prepared as described from yeast strain NOY384 transformed with pEGTy3-1 (40). Highly-purified TFIIIC (oligobox B+ fraction) and Pol III (MonoQ fraction) were purified as described (6). Purified wildtype, recombinant proteins were quantified as active molecules in specifically initiating transcription (pol III) or specific DNA binding (TBP, Brf1, Bdp1, TFIIIC), as described or referenced (41). TBP and Bdp1 were fully active; Brf1 was ~20% active. Amounts of Brf1 refer to total protein. Recombinant split Brf1 and Bdp1 used in these experiments were shown to have transcription activities on supercoiled and linear templates singly and in combination as previously reported or as indicated in Results (data not shown). Wild-type and internally deleted Bdp1 proteins were C-terminally His6-tagged and purified under native conditions through Ni-NTA agarose, Bio-Rex 70 and Superose 12, as previously described for Bdp1(138-596) in Kumar et al. (33). Bdp1(224-487), Bdp1(1-352) and Bdp1(352-594) were N-His6-tagged and purified under native conditions through the Ni-NTA agarose step. Wild-type Brf1 (Nand CHis6 tagged) and Brf1 deletion proteins (N-His6 or His7-tagged) were purified under denaturing conditions on Ni-NTA agarose (and on Superose 6 for Brf1(1-282) and Brf1(284-596)), followed by step-wise dialysis out of urea as specified in (36,41). TBP was purified and quantified as described (11). Quantities of mutant Brf1 and Bdp1 are specified as fmol of protein determined by Coomassie staining against BSA standards on gels. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 11 In vitro integration into SNR6 targets-In vitro integration with wild-type proteins was performed as described previously (23) except where noted otherwise. Where added, TFIIIC (100 fmol) was complexed with 150 fmol target DNA for 10 min in integration reaction buffer prior to addition of TFIIIB (50,180 and 75 fmol TBP, Brf1, and Bdp1 protein, respectively). Reaction volumes were 25 or 50 μl as noted. TFIIIB components were allowed to form complexes with target DNA for 60 min at 23C. At the end of this time, components were shifted to 15°C, 2.2 or 5 μg (protein) of Ty3 VLP fraction (depending on activity) was added, and the incubation was allowed to proceed for 10-15 min. Reaction samples were treated with proteinase K and extracted with phenol-chloroform. DNA was precipitated with ethanol. PCR with primer 242, which anneals within the SNR6 gene, and with primer 411, which anneals at the downstream end of the internal domain of Ty3, was used to amplify diagnostic fragments from one-fifth of the integration reaction volume (23). Reactions were initiated with a 95C polymerase activation step for 12 min, followed by 40 cycles of 95C for 30 s, 62C for 30 s, and 72C for 60 s. The 72C elongation step was extended by 3 s per cycle. The reaction was terminated with a 72C incubation for 5 min, after which the sample was brought to 4C. To control for consistent DNA recovery from the integration reaction and for consistent operation of the above PCR, primers 679 and 680 (23) were used to amplify the beta-lactamase gene carried by the target plasmid (data not shown). This PCR amplification was performed with 0.03% of the content of each integration reaction and with 200 ng of each primer for 19 cycles of polymerization. PCR products were resolved by by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 12 electrophoresis on a non-denaturing 8% polyacrylamide gel and visualized by staining with ethidium bromide. Integration reactions using SNR6 targets and mutant TFIIIB proteins-For integration reactions with the split Brf1 proteins and Brf1 ∆1-68∆383-424, Brf ∆383424, wild-type Brf1 (1 pmol), TBP (200 fmol), and Bdp1 (200 fmol) were used in 25 μl reactions. Each of the mutant Brf proteins was used at 200 fmol. Integration reactions containing Bdp1 half proteins were performed with 150 fmol of Bdp1, 200 fmol TBP and 1 pmol wild-type Brf1 in a 25 μl reaction. For Bdp1 internal deletion proteins and Bdp1(224-487), 25 or 50 fmol mutant protein was used, as indicated for specific experiments, in combination with 50 fmol TBP and 180 fmol Brf1 in a 25 μl reaction. Integration ladder--In vitro integration events into a SNR6 target plasmid were amplified as described above using primers 242 and 411. PCR reaction product DNA was digested with restriction enzymes XhoI and NruI for 1 hr at 37°C. XhoI cleaves within Ty3, 19 bp upstream of the site of integration on the non-transcribed strand; NruI cleaves within SNR6, 5 bp downstream of the start site of transcription. Integration downstream of bp +3 on the non-transcribed strand would not be monitored in this assay. Following digestion, reaction mixtures were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol. DNA pellets were resuspended in buffered formamide containing tracking dyes. One-third and two-thirds of the sample was used to examine the l-U6 and the r-U6 integration target sites, respectively. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 13 A sequencing ladder was generated using the method of Sanger (42) with 5'P-end-labeled primer 242 and pLY1855 or pU6LboxB template. Digested DNA from PCR reactions and the sequence ladder fragments were resolved on an 8%, 8M urea sequencing gel. Regions of the gel containing the digested integration products were transferred to nitrocellulose membrane using a semi-dry transfer apparatus, UV cross-linked, and PCR products were visualized by hybridization with 5' end-labeled oligo 451 which is complementary to the plus strand at the downstream (U5) end of the Ty3 element and exposed to a phosphor image screen. The length of the hybridized fragment estimated from the sequencing ladder allowed inference of the distance of the Ty3 strand transfer position on the nontranscribed strand from the transcription initiation site. RESULTS The amino-terminal half of Brf1 contains primary determinants of Ty3 integration into the SNR6 gene--B', comprised of the TFIIIB subunits Brf1 and TBP, was previously shown to be sufficient to mediate a low level of specific Ty3 integration. Of these two subunits, only Brf1 is specific to the pol III transcription initiation complex, suggesting that it may contribute directly to Ty3 targeting. In the case of pol III transcription on the SNR6 template pU6LboxB, it has been shown that the amino-terminal half of Brf1 (amino acids 1-282) supports transcription in the context of TFIIIB on supercoiled, but not linear templates (37). In contrast, the carboxyl-terminal half of Brf1 forms stable TFIIIB-DNA complexes, but is transcriptionally nearly inactive on supercoiled DNA (41). The amino-terminal and by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 14 carboxyl-terminal Brf1 half proteins together reconstitute transcription of linear and supercoiled DNA. In order to better define the domains of Brf1 involved in Ty3 targeting, integration reactions were performed using half Brf1 proteins in which the TFIIB-like and conserved carboxyl-terminal domains could be evaluated separately. Complete recombinant Brf1 and combinations of proteins representing the aminoterminal segments of Brf1 (1282 or 1-383) and carboxyl-terminal segments (284596 or 425-596) were used alone or as combined aminoand carboxyl-terminal parts. Supercoiled and linear target DNAs were evaluated. Incubation of supercoiled target DNA with TFIIIB for 60 min at 23C followed by addition of VLPs and 10 min incubation at 15C left 50% of the DNA supercoiled (data not shown). Because Ty3 integration in the absence of Bdp1 is significantly less efficient, these reactions were performed in the presence of Bdp1. Accordingly, these experiments address the relative contributions of different Brf1 domains, but not the minimum requirements for Ty3 integration. Reaction mixtures contained recombinant TBP, Bdp1, mutant Brf1 and plasmid pLY1855. The pLY1855 plasmid is a derivative of pU6LboxB (23), which contains a modified SNR6 gene with altered flanking sequence and a gene-internal boxB promoter element optimally placed for TFIIIC binding. In this construct the TATA box is inverted. Although both leftward (l-U6) and rightward (r-U6) initiation sites are used, TBP is preferentially bound in the orientation that supports leftward transcription (22). After completion of the incubation, reaction samples were extracted and processed for PCR, using primers to amplify target DNA containing by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 15 Ty3 insertions. The products of the PCR reaction were fractionated by gel electrophoresis in nondenaturing polyacrylamide gels, stained with ethidium and photographed (Fig. 1). As noted previously (23), two TFIIIB-dependent PCR products were generated on this template (lanes 2 and 12; compare with lanes lacking one or more components necessary for de novo integration: 1, 9-11, 19 and 20). The upper and lower bands represent integration into the l-U6 and r-U6 initiation sites, respectively. Although the relative yield of PCR product between reactions of a given experiment was reproducible, this assay should be considered semiquantitative, since it was not possible to accurately estimate the relative specific activities of the mutant proteins. In the absence of either Brf or TFIIIB, a somewhat random and dispersed background of integration events was observed (lanes 1, 10, and 11). Brf1(1-282) and Brf1(1-383) supported specific integration on supercoiled DNA and a greater amount of specific integration on linear DNA targets (lanes 3, 6, 13 and 16; this is most readily apparent at the l-U6 initiation site which is less obscured by the TFIIIB-independent background). The Brf1(284-596) and Brf1(425596) C-terminal segments failed to support specific integration on supercoiled targets (lanes 4 and 7) at a level significantly above background, but they greatly enhanced integration when combined with Brf1(1-282) and Brf1(1-383), respectively, (lanes 5 and 8). Brf1(425-596) likewise failed to support significant specific integration on a linear target (lane 17), but integration above background was detected with Brf1(284596) (lane 14). Thus, the portion of Brf1 containing the TFIIB-related putative zinc ribbon, two TFIIB-like repeats and the primary pol III interaction domain bears a major determinant for position-specific integration in a reaction also containing TBP, by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 16 Bdp1 and DNA. The Brf1 segment from amino acid 284 to 424 which contains fungal homology region l may also contain a determinant for specific integration. The zinc ribbon domain of Brf1 is not required for Ty3 integration into SNR6-Two motifs in the amino-terminal domain of Brf1 contribute to initiation of transcription: a putative amino-terminal zinc ribbon domain and the two TFIIB-related imperfect repeats. Disruption or removal of the zinc ribbon domain of Brf1 generates TFIIIB-DNA complexes that recruit pol III to relaxed DNA templates but display a severe defect in open complex formation. Combination with promoter openingdefective Bdp1 deletions eliminates transcription on supercoiled DNA templates as well (43,44,5). The N-terminal zinc ribbon also appears to be essential for transcription in the minimal pol III transcription system consisting of pol III, Brf1, TBP and a "pre-opened" promoter template (36). The ∆383-424 deletion, which removes sequence that is not present among fungal homologues, improves the transcriptional activity of Brf1 in vitro (36). In order to determine whether Ty3 integration is sensitive to the function provided by the zinc ribbon, recombinant Brf1 lacking the aminoterminal 68 amino acids containing the zinc ribbon and amino acids 383-424 (N∆68,∆383-424), and Brf1 lacking only the internal domain (∆383-424) were tested for ability to support integration (Fig. 2). Integration was supported by both of these deletion proteins to comparable extent on supercoiled and linear DNA (Fig. 2, compare lanes 2 and 5 with lanes 3 and 6). Bdp1 halves are redundant for enhancement of Ty3 integration into a TFIIIBDNA target--The role of Bdp1 in transcription complex formation appears to include a by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 17 scaffolding function that locks the complex together (4,33). Binding of Bdp1 to the B'DNA complex is accompanied by an upstream extension of the DNase I footprint, the introduction of an additional bend between the TATA box and the initiation site, and stabilization of the protein-DNA complex. Nevertheless, deletion analysis has so far failed to identify any specific portion of the protein that is essential for the extended footprint (33). Although B’ alone is sufficient to support Ty3 integration at SNR6 at a low level, addition of Bdp1 increases integration, suggesting that Bdp1 introduces additional contacts for the Ty3 preintegration complex, stabilizes the target complex, or changes its conformation (23). In order to more specifically define the requirement for Bdp1, recombinant Bdp1(1-352) and Bdp1(352-594) were tested together and separately for activity in Ty3 integration. These proteins support transcription of supercoiled SNR6 templates together and separately. Bdp1(1-352) and Bdp1(352-594) both supported integration into supercoiled DNA (Fig. 3, compare lanes 3 and 4 with lane 1). In addition, Bdp1 split proteins were tested in reactions with linear DNA. These reactions showed that on linear DNA the half and combined proteins also performed comparably to the wild-type Bdp1 (Fig. 3, compare lanes 10 and 11 with lane 9). These results suggest that Ty3 integration, similar to pol III transcription, is not dependent upon Bdp1 for a single contact. Either Bdp1 must have a structural role that does not involve specific contacts, or each part of Bdp1 individually provides a contact that makes the other part nonessential. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 18 TFIIIC interacts with B' and Bdp1 to influence Ty3 integration site selection-TFIIIC is required for SNR6 transcription and Ty3 integration in vivo (45,46,2). Although TFIIIC is dispensable for SNR6 transcription in vitro with purified components, TFIIIC-mediated assembly of TFIIIB onto the SNR6 TATA box specifies a single orientation of TFIIIB for transcription (22). Analysis of Bdp1 function in vitro has shown that TFIIIC-dependent transcription exhibits greater dependence upon requires functions in Bdp1 not required for TFIIIC-independent transcription (33). In particular, certain Bdp1 deletion mutants that are permissive for TFIIIC-independent transcription of supercoiled DNA, assemble aberrant TFIIIB-TFIIIC-DNA complexes on TFIIIC-dependent promoters that are transcriptionally deficient, or fail (entirely) to assemble these complexes. The experiments that are described next used supercoiled and linear DNA to explore the effect of TFIIIC on Ty3 integration at SNR6 directed by B' and also by TFIIIB constituted with wild-type Bdp1 or internal deletion mutants of Bdp1. The effect of TFIIIC on Ty3 integration directed by B' and TFIIIB was examined first (Fig. 4). There was no major difference in the distribution of integration sites between supercoiled and linear templates (compare Fig. 4A, lanes 14 with lanes 5-8). As previously observed (23), integration in the presence of B' alone was primarily into the l-U6 initiation site (lanes 1 and 5), whereas integration in the presence of TFIIIB was more evenly distributed between the l-U6 and r-U6 sites (lanes 3 and 7). This difference has been ascribed to weaker DNA-binding of the B' complex, which allows equilibration toward the optimum orientation of the B' complex by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 19 at the TATA box whereas entry of Bdp1 into the B' complex prevents dissociation, trapping the initial orientation of the B' complex. As previously shown (23), integration in the presence of TFIIIC and TFIIIB showed a dramatic shift to the r-U6 initiation site (lanes 4 and 8), consistent with TFIIIC orienting TFIIIB to favor initiation of r-U6 transcription into the U6 gene. In contrast, integration in the presence of TFIIIC and B' generated a small decrease in l-U6 integration on the supercoiled template and greater decrease in integration on the linear template, but did not show the dramatic increase in r-U6 integration shown in the presence of Bdp1 (compare lanes 1 and 5 with lanes 2 and 6). This result could be interpreted to suggest that TFIIIC does not affect the orientation of B' in the absence of Bdp1, but since entry of Bdp1 is dependent on the prior formation of the B'-TFIIIC-DNA complex (47), this is unlikely. The presence of significant integration at l-U6 may indicate that not all the templates contain B’ and TFIIIC. The absence of integration at r-U6 could stem from the fact that DNA surrounding the start site of transcription in B'-TFIIIC-DNA complexes is occluded by TFIIIC from attack by integrase; in the case of transcription, Bdp1 is required to lift TFIIIC from this site for transcription intiation to occur (47). The additional decrease in l-U6 integration observed here in the presence of Bdp1 (compare lanes 2 and 6 to lanes 4 and 8) could reflect the greater stability of the TFIIIB-TFIIIC complex compared to the B’-TFIIIC complex, trapping more of the target in this form. The site of Ty3 integration in vivo is precisely defined. It was of interest to determine whether the specificity of integration was conserved in vitro in the context by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 20 of minimal integration targets. In order to gain information concerning the overall distribution of integration sites, PCR products were digested with XhoI and NruI to remove both DNA ends, leaving an internal fragment the size of which was proportional to the distance of the integration site from the duplicated SNR6 transcription initiation sites (see Methods). These fragments were fractionated by electrophoresis on sequencing gels, transferred to nitrocellulose (48) and probed with a P-labeled oligonucleotide that anneals to the end of the Ty3 element in order to visualize only one DNA strand. The PCR products of integration into the l-U6 and rU6 transcriptional initiation sites separated into slower (I-U6)and faster (r-U6)migrating sets of fragments. Locations of integration sites were deduced from sequencing ladders and from parallel experiments with positive control plasmids pDLC370 and pLY1842, containing sequenced sites of integration at l-U6 and r-U6 (Fig. 4B). The results of this analysis showed that integration in the presence of B’ or TFIIIB occurred at one major site for l-U6 (Fig. 4C, lanes 1-8) and at two sites for rU6 (Fig. 4D, lanes 1-3 and 5-7). [Note that the relative amount of l-U6 and r-U6 radioactivity does not reflect the distribution of integration events, since different amounts of PCR product were loaded on each gel to obtain nearly equivalent radioactive signals.] Fragments generated in the restriction digestion of the PCR reaction were offset by one nucleotide from the major in vivo site of integration on the positive control r-U6 plasmid, but corresponded to sites that are also used in vivo. Integration sites were also mapped to identify the effects of TFIIIC on Ty3 integration site usage in the presence of B' and TFIIIB (Figs. 4C and D). There was no by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 21 redistribution of residual integration sites at l-U6 due to TFIIIC. TFIIIC also had no effect on the pattern of integration at r-U6 in the presence of B'. However, the pattern of integration at r-U6 in the presence of TFIIIC and TFIIIB was dramatically different from that of TFIIIB alone, with integration sites spread downstream into SNR6, from –7/-3 to –1/+4. This pattern contrasted with the positions of sites observed in vivo (predominantly at position -6/-2 and -7/-3). Mutations in Bdp1 that affect open complex formation do not affect Ty3 integration--The roles of specific Bdp1 domains in TFIIIC-dependent and TFIIICindependent integration can be further defined using Bdp1 internal deletion mutants. Analysis of the effect of a set of such mutants on pol III transcription in vitro (4,33,44) identified an internal segment defined by mutants Bdp1∆355-372, ∆372-387, ∆388409 and ∆409-421, within which deletions do not eliminate the ability of TFIIIB to recruit polymerase, but do interfere with formation of the open promoter complex. This domain is thus implicated either in isomerization of the polymerase or in DNA duplex destabilization (37). DNA structure has been found to affect integration activity of retroviral integrases (49-51). Thus, Bdp1 containing a deletion within this defined region offered an interesting in vitro test of the potential role of Bdp1 in creating a specific structure required by the Ty3 integrase for activity. Bdp1∆355372 was tested on linear and supercoiled SNR6 targets. It stimulated integration well over the levels observed with B' alone on both templates, with no significant change in distribution between l-U6 and r-U6 initiation sites (Fig. 5, lanes 4 and 12 relative to lanes 1 and 9). by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 22 Two domains of Bdp1 (I and II) are protected from hydroxyl radicals upon entry into theTFIIIB-SUP4 complex (33). Interactions involving domains I and II are required on an either-or basis for TFIIIC-independent transcription, but both are required for TFIIIC-dependent transcription. Bdp1 deletions in these domains were used to test whether domain I or II was required for the Bdp1 enhancement of specific integration over activity of B' alone in the absence of TFIIIC. Bdp1∆272-292 and Bdp1∆ 424-438, representing deletions in regions II and I, respectively, were shown to be as active as wild-type Bdp1 for TFIIIC-independent integration into linear and supercoiled SNR6 gene targets (Fig. 5, compare lanes 3, 5 with 2 and lanes 11 and 13 with 10). The finding that domains I and II of Bdp1 were not individually required for TFIIIC-independent integration is congruent with prior analysis of transcription. Bdp1∆424-438 fails in TFIIIC-dependent transcription because it does not assemble into the B'-TFIIIC-DNA complex. Bdp1∆272-292 assembles into the B'TFIIIC-DNA complex, but fails in TFIIIC-dependent transcription because it does not displace TFIIIC from the initiation site so as to allow pol III access (33). The effects of wild type Bdp1, Bdp1∆424-438 and Bdp1∆272-292 on Ty3 integration in the presence of TFIIIC were compared in order to examine whether TFIIIC would prevent integration by virtue of start site occlusion, or whether the presence of Bdp1 in the TFIIIB complex and integrase together would suffice for TFIIIC displacement (Fig. 6). Integration in the presence of wild-type TFIIIB alone produced more integration into the l-U6 than into the r-U6 initiation site (lane 1). As expected, TFIIIC by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Last printed 04/30/02 9:19 PM 23 redistributed this pattern to favor the r-U6 initiation site with new sites of integration downstream (Fig. 6A and B, lanes 2). Redistribution did not occur in reactions containing Bdp1∆272-292 which assembles into the B'-TFIIIC-DNA complex (lane 3) or, as expected, in the presence of Bdp1∆424-438 (lane 5), which does not assemble into a B'-TFIIIC-DNA complex. The core amino acid 224-487 fragment, which retains competence for TFIIIC-dependent transcription (33), yielded less integration, but significantly redistributed integration sites in response to TFIIIC (Fig. 6B, compare lanes 1 and 6). The observation that the presence of TFIIIC generates unique sites of integration at r-U6 (Fig. 4D) clarifies the analysis of TFIIIC effects on integration (Fig. 6B), because it substitutes a qualitative effect for a quantitative assessment that is burdened with a substantial background. TFIIIC generated downstream integration events with TFIIIB-DNA complexes containing wild type Bdp1, Bdp1∆355-372 and Bdp1(224-487) (compare lanes 2, 4 and 6 with lane 1), but not with TFIIIB-DNA complexes containing Bdp1∆272-292 (lane 3) or Bdp1∆424-438 (lane 5). These results imply a requirement for Bdp1-mediated displacement of TFIIIC from the site of Ty3 integration.