Functional interactions of DNA topoisomerases with a human replication origin

Abstract

The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.

Figure 1

Interaction of topo I and II with the lamin B2 origin in vivo. (A) LM-PCR-mediated analysis of the topo I–DNA cleavage complexes induced by 1 min treatment of asynchronously growing HeLa cells with increasing concentrations of CPT (1, 10 and 100 nM, lanes 3–5 and 1 μM, lanes 6 and 12) or with 10 μM gimatecan (lane 7). Lanes 2 and 11: control genomic DNA from untreated cells; lanes 1 and 10: Maxam–Gilbert sequencing reaction. TD-PCR analysis of the distribution of UV photoproducts along the origin region in UV-irradiated HeLa cells treated for 1 min with 1 μM CPT (lanes 9 and 14) or left untreated (lanes 8 and 13). (B) TD-PCR-mediated analysis of the topo II–DNA cleavage complexes induced by treatment of asynchronously growing HeLa cells with 10 nM VP16 (lanes 3 and 10); lanes 2 and 9: control genomic DNA from untreated cells; DNA immunoprecipitated with anti-topo II antibody from cells subjected (lane 4) or not subjected (lane 5) to VP16 treatment; TD-PCR analysis of the distribution of UV photoproducts along the origin region in UV-irradiated HeLa cells treated for 1 min with 10 nM VP16 (lanes 7 and 12) or left untreated (lanes 6 and 11). (C) Summary of topo I and II in vivo cleavage sites at the lamin B2 origin area involved in the replicative complexes. Leading strand start sites are indicated by arrows and DNA cleavage complexes by filled triangles.

Figure 2

Interaction of topo I and II with the lamin B2 origin in vitro. (A) Detection of the in vitro topo I cleavages stabilized on the lower strand by CPT (lane 3), 7-[CH2–Tris] CPT (lane 5) or gimatecan (lane 6), and on the upper strand by CPT (lane 10); lanes 7 and 12: Maxam–Gilbert sequencing reactions; the position of the cleavages also present in vivo is indicated by an asterisk. (B) Effect of base substitution mutations in the lamin B2 origin on topo I-mediated cleavage. (C) Sequence of the origin portion covered by the replicative complexes; the position of substituted bases is highlighted; the position of in vitro topo I-cleavable complexes is indicated by filled triangles; the asterisks indicate the position of the topo I cleavages also present in vivo. (D) Detection of the in vitro VP16-induced topo II cleavages introduced by the enzyme alone (lanes 1–5) or by topo II as part of a complex with nuclear proteins (lanes 6–14); lane 9: the origin DNA incubated with the nuclear extract and VP16 was immunopurified using anti-topo II antibody; black vertical bars indicate the region protected in vivo; the arrows indicate the borders of the region protected in vitro by the origin binding proteins (OBP) as determined by λ-exonuclease digestion; lanes 10 and 14: Maxam–Gilbert sequencing reactions.

Figure 3

Topo I interacts with the lamin B2 origin in a cell-cycle-dependent manner, and is a member of the origin binding complex. (A) Localization and orientation of the primer sets in the analyzed region; the positions of the detected topo I–DNA complexes are indicated by vertical arrows. (B) LM-PCR-mediated detection of CPT-induced topo I cleavage complexes on the lower and upper strands in different moments of the cell cycle; G, in vitro DMS-treated genomic DNA. (C) Identification of the presence of topo I on the CPT-induced cleavage sites and interaction of the enzyme with Orc2p; in the upper portion are shown the positions of the primers utilized (arrows) relative to topo I cutting sites; HeLa cells subjected to CPT treatment were crosslinked or not with DSP, lysed and the DNA was immunopurified with anti-topo I, anti-Orc2p or unrelated antibodies; the lower portion shows the PCR analysis of untreated genomic DNA (lanes 1 and 17), of the DNA immunopurified with anti-topo I antibodies (lanes 2–4), with anti-Orc2p antibodies (lanes 13–16) or with unrelated antibodies (lanes 5–12). (D) Topo I co-immunoprecipitates with Orc2p in HeLa nuclear extract: Western blot of proteins immunoprecipitated with anti-Orc2p antibody and assayed with anti-topo I or anti-Orc2p antibodies. (E) Formaldehyde crosslinking shows that both topo I and Orc2p associate with the lamin B2 origin in late G1; the DNA from formaldehyde crosslinked HeLa cells was immunopurified using anti-topo I or anti-Orc2 antibodies or pre-immune serum and subjected to competitive PCR analysis; B48, origin region; B13 non-origin region.

Figure 4

Topo II interacts with the lamin B2 origin in a cell-cycle-dependent manner, and is a member of the origin binding complex. (A) TD-PCR-mediated detection of VP16-induced topo II cleavages on the upper strand; G, in vitro DMS-treated genomic DNA. (B) Localization and orientation of the primer sets in the region analyzed; the positions of the detected topo II–DNA complexes are indicated by vertical arrows. (C) TD-PCR-mediated detection of VP16-induced topo II cleavages on the lower strand; G, in vitro DMS-treated genomic DNA. (D) TD-PCR analysis of DNA immunopurified with anti-Orc2p (lanes 3–6) or unrelated antibodies (lanes 1 and 2) from HeLa cells synchronized in the middle of G1, treated with VP16, DSP or both; topo II binding sites at or outside of the origin are indicated with one or two asterisks respectively; G, in vitro DMS-treated genomic DNA.

Figure 5

Inhibition of replicon activation following stabilization of topo I; 1. Stabilized topo I cleavage complexes at the lamin B2 origin do not lead to replication run-off. (A) Schematic representation of topo I-mediated single-strand breaks (SSB) induced by CPT (left side) or of the replication-dependent formation of double-strand breaks (DSB) (right side). (B) modified LM-PCR detection of the presence of SSB and DSB in the ori region; cells were collected at late G1 with mimosine, 1 μM CPT was added and they were released from mimosine for the indicated time; the same experiments were performed in the presence of 5 mM caffeine as indicated; DNA was isolated (see Materials and methods) and subjected to modified LM-PCR with (first two gels) or without (last two gels) first primer extension, as described (Strumberg et al, 2000). If no primers were added, no blunt-ended duplexes were formed, the linker ligation could not operate and no amplification was obtained, even in the presence of DNA polymerase added to complete possible recessed duplexes. If, after 30 min exposure to CPT, this drug is removed and the cells incubated for further10 min, the topo I-induced cut is completely abolished (last lanes of first two gels). The last gel shows the detection of double-stranded blunt ends produced by micrococcal nuclease treatment of cells or of naked DNA utilizing the modified LM-PCR procedure (Zaret, 2005) with primer set A.

Figure 6

Inhibition of replicon activation following stabilization of topo I; 2. Short nascent DNA of 0.6–1 kb was isolated by denaturing gel electrophoresis from HeLa cells collected at late G1 (mimosine synchronization) and pulse-labeled with BrdU in the presence of CPT (see Materials and methods). The top portion schematizes the expected distribution of BrdU-containing DNA (crosses and dots) if the origin fires in the absence (leftmost bubble) or presence of CPT (central bubble) or does not fire (rightmost bubble). The lower portion shows the quantification by competitive PCR analysis of the abundance of origin (B48) or non-origin (B13) sequences in the total nascent DNA and in DNA immunopurified with anti-BrdU antibody. Cells released from the mimosine block for 5 min enter S phase as shown by the comparable enrichment in origin (B48) over non-origin (B13) sequences of total and BrdU-labelled nascent DNA (left panel). Conversely, in the presence of CPT, the origin does not initiate, as shown by the absence of origin enrichment in the BrdU-containing DNA (the antibody efficiently precipitates nascent DNA molecules containing a single BrdU residue: Supplementary Figure 4).

Figure 7

Summary of the mapped protein–DNA interactions at the lamin B2 origin. The cartoon summarizes the interaction of active topo I and topo II molecules with the origin sequence along the cell cycle demonstrated in the present work and reports also the data previously obtained for the interactions of Orc1p, Orc2p and Cdc6p with the same sequence (Abdurashidova et al, 2003). The interaction of the two enzymes with the ORC complex is also shown.