Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication - PubMed (original) (raw)

Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication

H Laman et al. Mol Cell Biol. 2001 Jan.

Abstract

The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G(1)-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G(1)-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.

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Figures

FIG. 1

FIG. 1

Viral cyclins interact with DNA replication proteins. (A) Western analysis (WB) using JD32 antibody to detect in vitro-transcribed and translated human Orc1 and bacterially expressed GST-Orc1 fusion protein. (B) The indicated cyclins with N-terminal flag epitopes or an empty mammalian expression vector were transiently transfected into U2OS cells. Cell lysates were immunoprecipitated (IP) with anti-flag antibodies coupled to Sepharose and separated by SDS-PAGE, and Western analysis was performed with anti-Orc1 antibody (JD32). (C) Sf9 lysates from cells programmed to express the indicated cyclin-cdk complexes were used for kinase assays against the indicated GST fusion protein substrates. Kinase reaction products were resolved by SDS-PAGE, and 32P incorporation into the substrates was visualized and quantitated on a PhosphorImager.

FIG. 2

FIG. 2

Viral cyclin-cdk6 complexes functionally substitute for endogenous cdk2 activity. (A) Representative fields of nuclei replicating in vitro. Nuclei from HeLa cells arrested in late G1 phase by mimosine were incubated in replication buffer (buffer), supplemented with cytosolic extract from proliferating HeLa cells (+S20 extract), 0.5 mM roscovitine (RV), and recombinant cyclin-cdk complexes as indicated. Nuclear DNA was visualized by staining with propidium iodide (red signal), and DNA replicated in vitro was visualized by incorporation of biotin-dUTP and staining with streptavidin-FITC (green signal). Merged images of the two signals are presented, and nuclei replicating in vitro appears in yellow. (B) Quantitation of the percentages of nuclei replicating in these reactions. The percentages of nuclei (500 to 1,000 per reaction) replicating in the presence of the indicated compounds were quantitated from 6 to 10 independent experiments (except for the percentages in lanes labeled V and K, which were analyzed in two independent experiments). Mean values and standard deviations are presented. Lanes are as follows: buffer, buffer alone; S20, buffer plus S20 extract; RV, buffer plus S20 plus 0.5 mM roscovitine; then as indicated, cyclin-cdk complexes were added to roscovitine-treated samples. (C) Percentages of nuclei replicating in the presence of cycloheximide (chx). Mean values and standard deviations of two or three independent experiments performed in the presence of cycloheximide and the indicated compounds are as shown in panel B.

FIG. 3

FIG. 3

(A) V cyclin interacts with cdk4 and cdk6 but not cdk2. U2OS cells were transiently transfected with the indicated plasmids, and cell lysates were immunoprecipitated with antibodies to cyclins. Western analysis was performed on immunoprecipitates with the indicated anti-cdk antibodies and visualized by enhanced chemiluminescence detection. (B) K cyclin-cdk2 kinase activity is sensitive to roscovitine. Crude extracts from singly infected or coinfected Sf9 cells were used in kinase assays against a purified GST-pRB substrate in the presence or absence of roscovitine. Reaction products were resolved by SDS-PAGE, and 32P incorporation was analyzed by a PhosphorImager.

FIG. 4

FIG. 4

Viral cyclin-cdk complexes are sufficient to initiate replication in G1 nuclei when added to G1 cytosol. (A) Nuclei from NIH 3T3 cells isolated 17 h after release from quiescence were incubated in S or G1 cytosol supplemented with equivalent amounts of recombinant cyclin-cdk activity. In this and six other experiments with independent populations of nuclei and purified cyclins or cdk complexes, V cyclin-cdk6 and K cyclin-cdk6 consistently stimulated the proportion of nuclei capable of initiation in vitro. (B) Nuclei from 18-h NIH 3T3 cells were incubated in G1 cytosol supplemented with cyclin A-cdk2, V cyclin-cdk6, or K cyclin-cdk6, in combination with cyclin E-cdk2. In all cases cyclin E-cdk2 had a slight additional effect. (C) Images show representative populations of nuclei. Nuclei were scored using high-magnification fluorescence microscopy to detect the presence or absence of replication foci in 1 to 200 nuclei per sample. Nuclear DNA was visualized by staining with propidium iodide (red signal), and DNA replicated in vitro was visualized by incorporating dUTP-biotin as detected by streptavidin-FITC (green signal). Merged images of the two signals appear in yellow.

FIG. 5

FIG. 5

Viral cyclin-cdk6 complexes can initiate replication in trans in the presence of roscovitine. Nuclei from NIH 3T3 cells isolated 17 h after release from quiescence were incubated in G1 cytosol and recombinant cyclin-cdk complexes in the presence or absence of roscovitine (RV) as indicated. (A) Panels of nuclei incorporating dUTP-biotin as detected by streptavidin-FITC and viewed by confocal microscopy. (B) Inhibition of endogenous cdk2 activity with roscovitine prevents initiation by cyclin A-cdk2 complexes but not of viral cyclin-cdk6 complexes. A graph of the percentages of nuclei that continued to initiate replication in the presence of roscovitine is given. Data are representative of three separate experiments.

FIG. 6

FIG. 6

(A) Viral cyclins increase the proportion of cells in S/G2 phase. The indicated cyclins and a CD8 marker were transiently transfected into U2OS cells. At 48 h post transfection, the cells were harvested by trypsinization and stained with anti-CD8 antibodies conjugated to FITC. The cells were treated with propidium iodide and subjected to FACS analysis. Experiments 1 and 2 show the mean of triplicate samples, while experiments 3 and 4 were done in duplicate. (B) Representative histogram plots of CD8+ transfected cell populations as indicated. (C) Cells were transfected as in panel A, except that 24 h prior to harvesting and FACS analysis, 40 ng of nocodozole per ml was added. Numbers in the upper left hand corner are the percentages of cells in S phase. Results are representative of three independent experiments.

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