A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication

Abstract

In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.

Figure 1

Rb^1–400 inhibits origin unwinding after pre-RC formation. (A) Chromatin-loading assay. Sperm chromatin (10 000/μl) was incubated with 2 μl of HSS for 30 min and then supplemented with buffer (lane 1), 800 ng Rb^1–400 (lane 2), or 800 ng Rb^1–400 preincubated with 1.6 μg MBP-MCM7 peptide fusion (lane 3). After 30 min, 4 μl NPE was added, which contained aphidicolin (50 μg/ml) and buffer (lane 1), 400 ng Rb^1–400 (lane 2), or 400 ng Rb^1–400 and 800 ng MCM7 peptide (lane 3). After 45 min, the chromatin was purified, and bound proteins were analyzed by Western blot analysis using antibodies against RPA (lower panel), and a mixture of antibodies against MCM7, ORC2, and Cdc45 (upper panel). To measure DNA replication, the same reaction was carried out using NPE lacking aphidicolin but containing [α-³²P]dATP (lower panel). (B) DNA topology assay. Same as panel A, except that pBS (40 ng/μl) was used as the DNA template. Lane 2 shows the effect of p27^Kip addition. After 30 min incubation with NPE, the DNA was extracted, separated on a chloroquine agarose gel, and stained (top panel). To measure DNA replication, the same reaction was carried out using NPE lacking aphidicolin but containing [α-³²P]dATP (lower panel). (C) Sperm chromatin was incubated with HSS supplemented with control buffer (lanes 1 and 3), or 500 nM geminin (lane 2) for 30 min. Subsequently, buffer (lane 1), 800 ng Rb^1–400 (lane 2), or 800 ng Rb^1–400 preincubated with 1.6 μg MCM7 peptide (lane 3) was added. After further 30 min, chromatin-bound proteins were analyzed with antibodies against MCM4, Rb, or ORC2.

Figure 2

Rb^1–400 protein inhibits DNA replication and chromosome unwinding after initiation. (A) RPA loading in the presence of actinomycin D is Cdk2-dependent. Sperm chromatin was incubated with HSS, followed by unsupplemented NPE (lanes 1–4), or NPE containing 10 μM actinomycin D (lanes 5), actinomycin D and p27^Kip (lane 6), or aphidicolin (lane 7). At the indicated times, chromatin was isolated and blotted for MCM7, ORC2, Cdc45, and RPA. (B) Model for the actinomycin D and aphidicolin arrest points. (C) Sperm chromatin was incubated with HSS, followed by NPE^actD, and isolated (lane 1), or isolated and then incubated with buffer (lane 2), 800 ng Rb¹⁻⁴⁰⁰ (lane 3), or 800 ng Rb¹⁻⁴⁰⁰ preincubated with 1.6 μg MCM7 peptide. Isolation leads to permanent immobilization of the sperm on the tube. After 30 min, the supernatant was replaced with 5 μl fresh NPE^aph containing buffer (lane 2), 400 ng Rb¹⁻⁴⁰⁰ (lane 3), or 400 ng Rb¹⁻⁴⁰⁰ preincubated with 800 ng MCM7 peptide. After 45 min, chromatin was washed and blotted for MCM7, ORC2, Cdc45, and RPA34 (upper panel). Identical reactions were carried out in which the second incubation with NPE lacked aphidicolin but contained [α-³²P]dATP to measure DNA replication (bar graph).

Figure 3

Rb^1–400 inhibits DNA replication and chromosome unwinding during elongation. (A) DNA replication kinetics at 22 and 19°C. Sperm chromatin was pre-incubated with HSS, NPE was added, reactions were transferred to 22°C (squares) or 19°C (circles), and DNA replication was measured. (B) All origins fire within 20 min of NPE addition at 19°C. Sperm chromatin was incubated with HSS, followed by NPE addition and transfer to 19°C. Buffer (circles) or p27^Kip was added 0 min (squares) and 20 min (triangles) after NPE. (C) Rb^1–400 inhibits the elongation complex. Sperm chromatin was incubated with HSS, followed by addition of NPE and transfer to 19°C. After 25 min, p27^Kip was added, and after a further 15 min chromatin was isolated (lane 1). Chromatin was exposed to buffer (lane 2), Rb^1–400 (lane 3), or Rb^1–400/MCM7 (lane 4). Finally, fresh NPE containing aphidicolin, p27^Kip, and buffer (lane 2), Rb^1–400 (lane 3), or Rb^1–400/MCM7 peptide (lane 4) was added to measure chromatin binding (lower panel). To measure DNA replication (bar graph), aphidicolin was omitted and both NPEs contained [α-³²P]dATP.

Figure 4

Rb^1–400 inhibits DNA replication of chromatin containing low levels of MCM2–7. (A) Effect of HSS dilution on MCM2–7 loading. Sperm chromatin was incubated with HSS (lanes 1 and 2), or HSS that was diluted 10-fold with ELB (lanes 3 and 4), in the presence (lanes 2 and 4) and absence (lanes 1 and 3) of geminin. After 30 min, the chromatin was isolated and blotted for MCM7 and ORC2. (B) Effect of Rb¹⁻⁴⁰⁰ on chromatin containing low levels of MCM2–7. Sperm chromatin was incubated with HSS (1 ×) or 10 times diluted HSS (0.1 ×), some of which contained 500 nM geminin (lanes 3 and 4). Subsequently, NPE was added at 19°C, and after 25 min p27^Kip was added. After a further 15 min, the chromatin was isolated and exposed to buffer, Rb¹⁻⁴⁰⁰, or Rb¹⁻⁴⁰⁰/MCM7 peptide. Fresh NPE containing buffer, Rb¹⁻⁴⁰⁰ or Rb¹⁻⁴⁰⁰/MCM7 peptide was added, and DNA replication was measured after 45 min.

Figure 5

Cdc45 is required for chromosome unwinding during elongation. (A) Cdc45 is required for chromosome unwinding near origins. After incubation in HSS followed by NPE^actD, chromatin was isolated and stopped (lane 1), or isolated and exposed to buffer (lane 2), 0.4 μg/μl Cdc45 IgG (lane 3), or Cdc45 IgG that had been pre-incubated with 90 ng/μl purified His-tagged Cdc45 protein (lane 4). After 30 min, the chromatin was washed, and fresh NPE was added, which contained aphidicolin to measure chromatin loading of RPA or [α-³²P]dATP to measure DNA replication. (B) The same low-temperature synchronization approach used in Figure 3C was used, except that inhibition was carried out using Cdc45 antibodies as in (A), and DNA replication (bar graph), or chromatin loading of MCM7, ORC2, Cdc45, and RPA in the presence of aphidicolin (upper panel) was measured.

Figure 6

Cdc45 has a slow off-rate from chromatin. Sperm chromatin containing the activated helicase was generated and purified as in Figure 2C. Subsequently, it was incubated with mock-depleted (lane 1) or Cdc45-depleted NPE (lane 2). In the same experiment, sperm chromatin was incubated with mock-depleted HSS and NPE (lane 3) or Cdc45-depleted HSS and NPE (lane 4). At 45 min after the final NPE addition, DNA replication and chromatin loading in the presence of aphidicolin were measured.