Unphosphorylatable mutants of Cdc6 disrupt its nuclear export but still support DNA replication once per cell cycle.

Cdc6 is essential for eukaryotic DNA replication. We have mutated highly conserved CDK phosphorylation sites in Cdc6. Contrary to their reported phenotypes in human cells, unphosphorylatable DeltaCDK mutants fully support DNA replication in Xenopus eggs. WtCdc6 is actively exported from the nucleus, which could explain why nuclear permeabilization is required for reinitiation within one cell cycle. However, DeltaCDK mutants are retained in the nucleus, yet surprisingly they still support only one round of replication. As these highly conserved CDK sites are unnecessary for replication once per cell cycle, an alternative checkpoint role for monitoring completion of the S phase is suggested.

The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells.

Most eukaryotic cell types can withdraw from proliferative cell cycles and remain quiescent for extended periods. Intact nuclei isolated from quiescent murine NIH3T3 cells fail to replicate in vitro when incubated in Xenopus egg extracts, although intact nuclei from proliferating cells replicate well. Permeabilization of the nuclear envelope rescues the ability of quiescent nuclei to replicate in the extract. We show that origin replication complex (ORC), minichromosome maintenance (MCM), and Cdc6 proteins are all present in early quiescent cells. Immunodepletion of Cdc6 or the MCM complex from Xenopus egg extract inhibits replication of permeable, quiescent, but not proliferating, NIH3T3 nuclei. Immunoblotting results demonstrate that mouse homologues of Mcm2, Mcm5, and Cdc6 are displaced from chromatin in quiescent cells. However, this absence of chromatin-bound Cdc6 and MCM proteins from quiescent cells appears not to be due to the absence of ORC subunits as murine homologues of Orc1 and Orc2 remain chromatin-bound in quiescent cells. Surprisingly, intact quiescent nuclei fail to bind exogenously added XCdc6 or to replicate in Xenopus egg extracts immunodepleted of ORC, even though G1- or S-phase nuclei still replicate in these extracts. Our results identify Cdc6 and the MCM complex as essential replication components absent from quiescent chromatin due to nonfunctional chromatin-bound ORC proteins. These results can explain why quiescent mammalian nuclei are unable to replicate in vivo and in Xenopus egg extracts.

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process.

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1 )phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

Selection and mapping of replication origins from a 500-kb region of the human X chromosome and their relationship to gene expression.

In higher eukaryotes the mechanism controlling initiation of DNA replication remains largely unknown. New technologies are needed to shed light on how DNA replication initiates along the genome in specific regions. To identify the human DNA sequence requirements for initiation of replication, we developed a new method that allows selection of replication origins starting from large genomic regions of human DNA. We repeatedly isolated 15 new putative replication origins (PROs) from a human DNA region of 500 kb in which 17 genes have previously been characterized. Fine-mapping of these PROs showed that DNA replication can initiate at many specific points along actively transcribed DNA in the cell lines used for our selection. In conclusion, in this paper we describe a new method to identify PROs that suggests that the availability of initiation sites is dependent on the transcriptional state of the DNA.

Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction.

A procedure was developed for mapping origins of DNA replication in mammalian cell chromosomes based on determining the relative abundance of nascent DNA strands throughout a specific genomic region. The method entails purification of short strands of nascent DNA derived from recently activated origins and the quantification, within this sample, of the relative abundances of different adjacent DNA segments by a competitive polymerase chain reaction technique. It is expected that the abundance of defined markers within the origin region is greatest at the site where DNA replication begins. This origin mapping procedure (i) allows analysis of single-copy genomic regions, (ii) can be performed on cultured and primary cells in the absence of any chemical treatment, (iii) does not require cell synchronization, and (iv) allows mapping origins to within a few hundred base pairs. This high degree of resolution permits a study of the cis- and trans-acting elements required for origin function. Application of this method to single-copy sequences in mammalian cells has identified replication origins within an approximately 500-bp segment in the human lamin B2 gene domain and within an approximately 800-bp segment in the hamster dihydrofolate reductase gene locus.

High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.

By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.