Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication

Abstract

Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3'-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5' DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process. Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3' overhangs and also interacts with Tpg, bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA. Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates. They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization.

Figure 1

Tap ORFs in vicinity of terminal protein genes (tpg). (Top) Alignment of the amino acid sequences of Tap proteins in Streptomyces coelicolor (Tap_C), Streptomyces lividans (Tap_L), and Streptomyces rochei (Tap_R1, Tap_R2, and Tap_R3). TapR1-R3, Tap_R1 or Tap_R3; TapC-L, Tap_C or Tap_L. (A) Schematic map of gene arrangements of tpg and tap in S. rochei (Tap_R1,R2,R3 and TpgR1, 2, 3), S. lividans (Tap_L and TpgL), and S. coelicolor (Tap_C and TpgC). The number of nucleotides between the stop codon of the tap gene and the start codon of the tpg gene are shown in the boxes. (B) Reverse transcriptase (RT)–PCR analysis of tap and tpg in S. lividans 1326. (Lane 1) 1-kb DNA ladder. (Lane 2) RT–PCR product of the primer pair of RT–cDNA and RT_0.6kb. (Lane 3) RT–PCR product of the primer pair of RT–cDNA and RT_1.3kb. (Lane 4) RT–PCR product of the primer pair of RT–cDNA and RT_2.0kb. (Lane 5) RT–PCR product of the primer pair of RT–cDNA and RT_2.4kb. (Lane 6) RT–PCR product of the primer pair of RT–cDNA and RT_2.9kb.

Figure 2

DNA binding activity and footprinting analysis. (A) EMSA using radioactively labeled single-stranded pSLA2 telomeric DNA. (Lane a) Probe + BSA as control. (Lanes b,c,d) TpgL protein with 5-fold, 20-fold, and 100-fold excesses of single-stranded BKKO5 DNA. (Lanes e,g,f) Same as lanes b, c, and d except Tap_L protein was used. (B) EMSA using radioactively labeled single-stranded pSLA2 telomeric DNA in the presence of unlabeled cold probe as competitor. (Lane a) Probe + BSA as control. (Lanes b,c,d) TpgL protein with 5-fold, 20-fold, and 100-fold excesses of single-stranded cold probe. (Lanes e,g,f) Same as lanes b, c, and d except Tap_L protein was used. (C) EMSA using radioactively labeled double-stranded pSLA2 telomeric DNA. (Lane a) Probe + BSA as control. (Lanes b,c,d) TpgL protein with 5-fold, 20-fold, and 100-fold excesses of circular chromosomal DNA of BKKO5. (Lane e) Tap_L protein alone. (D) DNase I footprinting analysis using single-stranded pSLA2 telomeric DNA and Tap_L protein was carried out as described in Materials and Methods. (Lanes 1,2) Single-stranded telomeric DNA was incubated with 0 and 2 μg of Tap_L protein, respectively. (Lanes 3–6) DNA sequencing ladder reactions with termination mix of ddG, ddA, ddT, and ddC. Brackets at the right show two regions of the protected DNA sequences.

Figure 3

Tap coimmunoprecipitates with Tpg. (A–D) Streptomyces lividans 1326 cell extracts were incubated with prebleeding sera (lane 1), anti-TpgL antibodies (lane 2), and anti-Tap_L antibodies (lane 3). (A) Western blotting using anti-TpgL antibodies. (B) Immunodepletion analysis of lanes shown in A using anti-TpgL antibodies. (C) Western blotting using anti-Tap_L antibodies. (D) Immunodepletion analysis of lanes shown in C using anti-Tap_L antibodies. (E) Agarose gel electrophoresis of the products of IP–PCR using different immunoprecipitates as templates and the telomere-specific primers P-end and P300 (Materials and Methods). (Lane 1) 1-kb DNA ladder. (Lane 2) IP with prebleeding anti-sera as negative control. (Lane 3) IP with anti-TpgL. (Lane 4) IP with anti-Tap_L. (Lane 5) Template of total DNA of S. lividans 1326 as positive control.

Figure 4

Yeast two-hybrid analysis of Tap_L and TpgL. Selection plates of SC-Leu-Trp (A), SC-Leu-Trp-His + 3AT (B), X-gal (C), SC-Leu-Trp-Ura (D), and SC-Leu-Trp + 0.2% 5FOA (E) were used to examine reporter gene phenotypes of yeast strain MaV203. (Row 1) Cotransformants of pDBLeu and pDEST22 as negative control. (Row 2) Cotransformants of pPC97-Fos and pPC86-Jun as positive control. (Row 3) Cotransformants of pBC191 and pDEST22. (Row 4) Cotransformants of pBC193 and pDBLeu. (Row 5) Cotransformants of pBC191 and pBC193.

Figure 5

Analysis of Streptomyces lividans 1326 chromosomal DNA showing disruption of tap_L gene and both tap_L and tpgL. (Top) Schematic representation of gene disruption. tap_L was disrupted by an in-frame insertion of spc on pBC131; both tap_L and tpgL were disrupted by the replacement of spc on pBC141. am, apramycin-resistance gene; spc, spectinomycin-resistance gene. (A) Ethidium bromide-stained agarose gel. (Lane 1) 1-kb DNA ladder. (Lane 2) 1326 total DNA. (Lane 3) BKKO17 total DNA. (Lane 4) BKKO19 total DNA. (Lane 5) pBC117 plasmid DNA. (Lane 6) pBC141 plasmid DNA. (Lane 7) pBC129 plasmid DNA. (B) Southern blot of same gel probed with ³²P-labeled pBC141. All DNAs were digested with PstI.

Figure 6

Whole-genome comparison of knockout strain BKKO17 (tap_L⁻) or BKKO19 (tap_L⁻ tpgL⁻) with wild-type Streptomyces lividans 1326. Profiles indicate gene deletion and amplification in knockout strains. Genes were analyzed and clustered by GABRIEL analysis (Pan et al. 2002). Consistency in the amount of DNA used for analysis at different repeats was inferred from our finding that 97% of the genes analyzed showed no change in the compared strains. DNA ratios are represented in tabular form according to the color scale shown at the top; rows correspond to individual genes, and columns correspond to different repeats. Green shades represent gene deletion in knockout strain, and red shades represent gene amplification in knockout. Black indicates unchanged ORFs in the comparing DNA. jncL, junction of the left chromosome end; jncR, junction of the right chromosome end. Lanes 1–4 represent 4 repeats. (A) Profile of the comparison of BKKO17 and S. lividans 1326. TS1, 2, 3, and 4, telomere sequences 1, 2, 3, and 4. Chromosomal telomere DNA was PCR-amplified and printed on the DNA microarray slides for detecting telomere loss. (B) Profile of the comparison of BKKO19 and S. lividans 1326. (C) The ordered cosmids at two chromosomal ends (Redenbach et al. 1996). Green dots represent terminal protein covalently bound to chromosome end. Black arrows point to the chromosomal breakpoints.

Figure 7

Analysis of plasmid DNA replication of pSLA2 derivatives (pBC167, pBC178, and pBC181) in Streptomyces lividans 1326 and in the tap_L and tpgL double-knockout strain BKKO19 (with a circular chromosome). pBC167 contains a functional tpgL gene, pBC178 contains a functional tap_L gene, and pBC181 contains both tpgL and tap_L. Streptomyces linear plasmid DNA was isolated from 1326 and BKKO19 transformants by treatment with proteinase K and SDS (Qin and Cohen 1998, 2000) and was electrophoresed for 12 h at 30 V in a 0.6% agarose gel; linear samples indicated by + were isolated by an alkaline lysis procedure (incubation in 0.2 N NaOH at 37°C for 30 min) followed by phenol/chloroform extraction to remove or degrade linear plasmids and chromosomal DNA fragments (Qin and Cohen 1998, 2000). (Lane 1) 1-kb DNA ladder. (Lane 2) SspI-digested pBC167 plasmid DNA isolated from Escherichia coli. (Lane 3) DNA isolated from a 1326 transformant receiving SspI-cleaved pBC167. (Lane 4) Same DNA as in lane 3, but following NaOH treatment. (Lane 5) SspI-digested pBC178 plasmid DNA. (Lane 6) DNA isolated from a 1326 transformant receiving SspI-cleaved pBC178. (Lane 7) Same DNA as in lane 6, but following NaOH treatment. (Lane 8) SspI-digested pBC181 plasmid DNA. (Lane 9) DNA isolated from a 1326 transformant receiving SspI-cleaved pBC181. (Lane 10) Same DNA as in lane 9, but following NaOH treatment. (Lane 11) DNA isolated from a BKKO19 transformant receiving SspI-cleaved pBC181. (Lane 12) Same DNA as in lane 11, but following NaOH treatment. (Lane 13) The banding positions of chromosomal (chr) and plasmid (ccc, covalently closed circular; OC, open circular) DNAs isolated from BKKO19 transformants receiving uncleaved pBC178 DNA. (Lane 14) The bands that were recovered from lane 13 DNA following NaOH treatment.