RNAP-II molecules participate in the anchoring of the ORC to rDNA replication origins

Abstract

The replication of genomic DNA is limited to a single round per cell cycle. The first component, which recognises and remains bound to origins from recognition until activation and replication elongation, is the origin recognition complex. How origin recognition complex (ORC) proteins remain associated with chromatin throughout the cell cycle is not yet completely understood. Several genome-wide studies have undoubtedly demonstrated that RNA polymerase II (RNAP-II) binding sites overlap with replication origins and with the binding sites of the replication components. RNAP-II is no longer merely associated with transcription elongation. Several reports have demonstrated that RNAP-II molecules affect chromatin structure, transcription, mRNA processing, recombination and DNA repair, among others. Most of these activities have been reported to directly depend on the interaction of proteins with the C-terminal domain (CTD) of RNAP-II. Two-dimensional gels results and ChIP analysis presented herein suggest that stalled RNAP-II molecules bound to the rDNA chromatin participate in the anchoring of ORC proteins to origins during the G1 and S-phases. The results show that in the absence of RNAP-II, Orc1p, Orc2p and Cdc6p do not bind to origins. Moreover, co-immunoprecipitation experiments suggest that Ser2P-CTD and hypophosphorylated RNAP-II interact with Orc1p. In the context of rDNA, cryptic transcription by RNAP-II did not negatively interfere with DNA replication. However, the results indicate that RNAP-II is not necessary to maintain the binding of ORCs to the origins during metaphase. These findings highlight for the first time the potential importance of stalled RNAP-II in the regulation of DNA replication.

Figure 1. The replication of rDNA is affected in the absence of the largest subunit of RNAP-II.

(a) Diagram of the rDNA with the locations of the replication barrier (RFB), replication origin (ARS) and cohesin binding sequences (CAR). Below, the theoretical schemes for the two-dimensional agarose gel electrophoresis of chromatin digested with BglII are depicted. The accumulation of RIs at the RFB is expected at 1.49X. 1X represents the position of the linear, unreplicated fragment. The hybridisation probe used for the Southern protocol and hybridisation was amplified by PCR and column purified (Qiagen). The amplified fragment is represented above. The direction of the anticlockwise and clockwise replication fork is shown below. (b) Results obtained after the isolation of DNA from asynchronous cultures growing exponentially at different temperatures. Higher exposure images are shown below. Significantly fewer amounts of RIs were detected in the absence of RPB1 (rpb1–1 ts strain). 1X signals were similar among the 4 experiments (25°C, 37°C for 30 minutes, 37°C for 1 hour and 37°C for 3 hours). The same number of cells was analysed. (c) Results obtained after fob1 deletion in the rpb1–1 ts strain. (d) To control the effect of the temperature on DNA replication, a wild type strain (BY4741) was used. (e) Results obtained after the isolation of DNA from a fob1Δ strain containing approximately 190 or 25 rDNA copies. A higher exposure image is shown on the right for the 25 copies strain.

Figure 2. The inhibition of RNAP-II does not alter the replication of rDNA.

(a) rpb1–1 ts strain grown at 25°C. Exponentially growing cultures were treated with 10 µg/ml of AM for 1 hour or with 200 mM of DRB for 4 hours before cell harvesting as previously reported . The quantification of RNA was performed using real-time qRT-PCR. The RNA levels are shown relative to each primer (untreated). To compare the conditions, the data were normalised relative to the mature form of the U2 small nuclear RNA . A representative diagram of the rDNA gene locus on chromosome XII is shown below. Specialised features of the IGS include the RNAP-II promoter (green circles), cohesin binding sequence (CAR) (grey box), a replication origin (ARS) in IGS2 and a replication fork barrier (RFB) in IGS1. The locations of the primers used in this study are shown. All values are expressed as the mean ± S.E.M. n = 2, n = 3 or n = 4. **p<0.005, *p<0.05 for Student’s t-test, untreated versus treated. (b) 2D gels of the RIs corresponding to untreated and AM and DRB treated samples.

Figure 3. The inhibition of transcription by RNAP-II does not affect the binding of replication components to the rDNA locus.

(a) Chromatin immunoprecipitation (ChIP) analysis of RNAP-II (4H8), Orc1p, Orc2p and Cdc6p binding within the intergenic spacers (primers 15 to 22), 35S (primer 13 and 23) and 5S gene regions (primer 18) in wild type cells. Orc1p, Orc2p and Cdc6p bound to cohesin (CAR, primer 19 and 20) and ARSs (p20, p21). (b) ChIP analysis of Orc1p, Orc2p and Cdc6p bound to the ARS in a rpb1–1 ts strain growing at 25°C. The results were obtained for untreated and cells that were treated with AM for 1 hour or DRB for 4 hours. All values are expressed as the mean ± S.E.M. n = 2 or n = 3. **p<0.005, *p<0.05 for the Student’s t-test, untreated versus treated. (c) ChIP analysis of the rpb1–1 ts strain growing at 25°C or shifted at 37°C for 30 minutes. Mean ± S.E.M. n = 3 or n = 4. **p<0.005, *p<0.05 for Student’s t-test, 25°C versus 37°C. A sequence located in chromosome VI was used as a negative control (background) for the binding of the replication proteins.

Figure 4. RNAP-II participates in the anchoring of Orc1p, Orc2p and Cdc6p to the ARS.

(a) 2D gel of the RIs corresponding to an asynchronous culture (rpb1–1 strain) that was arrested with α-factor in G1 at 25°C and subsequently was divided into four aliquots. α-factor at 25°C and α-factor at 37°C for 45 additional minutes: Two flasks were released with pronase, and the cells were grown at 25°C or shifted to 37°C for 45 minutes. (b) ChIP analysis of Orc1p, Orc2p and Cdc6p within the IGS regions in rpb1–1 cells arrested in G1 at 25°C and shifted to 37°C. On the right is the ChIP analysis obtained after releasing cells 45 minutes after pronase at 25°C or 37°C. (c) and (d) ChIP analysis and images corresponding to a 2D gel obtained from an asynchronous culture (wild type) that was arrested in G1 with α-factor and subsequently divided into four aliquots as described above. (e) and (f) ChIP analysis and 2D gels show the RIs of the asynchronous culture (wild type) used to arrest cells in G1 (α-factor). The cells were incubated for 45 additional minutes in the presence or absence of 10 µg/ml of AM. The cells that were released from α-factor were incubated in the presence or absence of AM. The fold enrichment relative to a sequence located in ChVI is shown. Mean ± S.E.M. n = 2 or n = 3. **p<0.005, *p<0.05 for Student’s t-test, 25°C versus 37°C, untreated versus treated.

Figure 5. Co-immunoprecipitation experiments suggest that RNAP-II complexes interact with Orc1p.

(a) Western-blot analysis using 4H8 antibody for immunoprecipitated Orc1p, Orc2p, Cdc6p and RNAPII (using the 4H8, 8GW16 or H5 antibody). Hypophosphorylated (8GW16) and Ser2P-CTD (H5) phosphorylated at Ser5 (4H8). The same number of cells was used for each immunoprecipitation experiments (see materials and methods). Western-blot analysis using Orc2p or Cdc6p antibody, showed the interaction between Orc2p and Cdc6p. NO (no antibody) (b) Western-blot using Orc1p antibody for immunoprecipitation revealed that Ser2P-RNAP-II (H5) interacts with Orc1p. No interaction of RNAP-II with Orc2p was detected when Orc2p antibody was used for the western-blot (c) Co-IP results show that hypophosphorylated RNAP-II complexes (8GW16) interact with Orc1p, 4H8 and H5. The results for whole cell extraction (WCE) and immunoprecipitation performed without antibody (NO) are shown.

Figure 6. The levels of RNAP-II bound to the chromatin affects DNA replication at the rDNA locus.

(a) ChIP analysis shows the binding of Ser2P-CTD to the rDNA before and after treatment with AM and DRB. Values are expressed as the mean±S.E.M. n = 2. (b) ChIP analysis of Ser2P-CTD (H5) and Ser5P-CTD (4H8) using a wild type strain and sir2Δ within rDNA IGS regions. Mean ± S.E.M. n = 3. *p<0.05 for Student’s t-test, wild type versus sir2Δ. (c) rtPCR-based analysis of transcripts within IGS regions in wild-type and sir2Δ. Mean ± S.E.M. n = 3. **p<0.005, *p<0.05 for Student’s t-test, wild type versus sir2Δ. (d) ChIP analysis of RNAP-II (4H8 and H5) binding in a GAL-REB1 strain. The cells were grown in a 2% galactose/0.3% glucose medium (+REB1) and shifted to 2% glucose to repress REB1 expression (REB1-). An RT-PCR-based analysis of the IGS transcripts is shown on the right. Mean ± S.E.M. n = 2 o n = 3. **p<0.005, *p<0.05 for Student’s t-test, REB+ versus REB- (7 generations). (e) 3C analysis in a GAL-REB1 strain. Controls for random ligation (R2+F4; [38]) are shown. Mean ± S.E.M. n = 3. *p<0.05 for Student’s t-test, REB1+ versus REB1-. (f) 2D gels of RIs corresponding to sir2Δ and GAL-REB1 strain grown in a 2% galactose/0.3% glucose medium (REB1+) or in 2% glucose for 5 or 7 generations (REB-).

Figure 7. Rpb1p does not participate in the binding of ORC complex to replication origins in nocodazole arrest.

(a) Schematic representation of the ORIs located at different chromosomes. ChIP analysis of Orc1p and Orc2p to ARS604, ARS1412 and ARS607 in rpb1–1 strain at 25°C and shifted at 37°C for 45 minutes. Mean ± S.E.M. n = 3. *p<0.05 for Student’s t-test, 25°C versus 37°C. (b) ChIP analysis of Orc1p and Orc2p within the rDNA IGS regions of chromosome XII and ARSs located in different chromosomes in rpb1–1 cells arrested in metaphase at 25°C (with nocodazole) and shifted to 37°C for 45 additional minutes. A chromosome VI sequence was used as a negative control for the binding of the replication proteins. Mean ± S.E.M. n = 3.