Mark A. Madine

The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin

BACKGROUND The origin recognition complex (ORC) and the minichromosome maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential for DNA replication. Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus. The Xenopus MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc 1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essential for DNA replication. Here, we describe the different but interdependent functions of the ORC and MCM complexes in DNA replication in Xenopus egg extracts. RESULTS The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts. Immunodepletion of ORC inhibits DNA replication of Xenopus sperm nuclei. Mixing MCM-depleted and ORC-depleted extracts restores replication capacity. ORC does not co-localize with sites of DNA replication during elongation. However, at initiation the two staining patterns overlap. In contrast to MCMs, which are displaced from chromatin during S phase, XOrc1 and XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis. Permeable HeLa G1- and G2-phase nuclei can replicate in ORC-depleted extract, consistent with the presence of chromatin-bound ORC in both pre-replicative and post-replicative nuclei. Interestingly, the binding of ORC to chromatin does not require the presence of MCMs; however, the binding of MCM proteins to chromatin is dependent on the presence of ORC. CONCLUSIONS The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S. cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis. The binding of MCM proteins requires the presence of ORC. Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA.

A rotary pumping model for helicase function of MCM proteins at a distance from replication forks

We propose an integrated model for eukaryotic DNA replication to explain the following problems: (1) How is DNA spooled through fixed sites of replication? (2) What and where are the helicases that unwind replicating DNA? (3) Why are the best candidates for replicative helicases, namely mini‐chromosome maintenance (MCM) proteins, not concentrated at the replication fork? (4) How do MCM proteins spread away from loading sites at origins of replication? We draw on recent discoveries to argue that the MCM hexameric ring is a rotary motor that pumps DNA along its helical axis by simple rotation, such that the movement resembles that of a threaded bolt through a nut, and we propose that MCM proteins act at a distance from the replication fork to unwind DNA. This model would place DNA replication in a growing list of processes, such as recombination and virus packaging, that are mediated by ring‐shaped ATPases pumping DNA by helical rotation.

Interaction of Xenopus Cdc2·Cyclin A1 with the Origin Recognition Complex

The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G1/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation ofXenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21Cip1. This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2·cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.

Xenopus replication assays.

Publisher Summary This chapter discusses the xenopus replication assays. It describes the preparation and use of both Xenopus egg extracts and templates for in vitro DNA replication. This chapter outlines a range of methods commonly employed to assay DNA replication. Eggs of Xenopus laevis, and extracts derived from eggs, are capable of assembling purified DNA or chromatin into functional nuclei that then undergo a single complete round of DNA replication. Noticeably, efficient replication is observed only when DNA is assembled into nuclei. Unless restrained by protein synthesis inhibitors, such as cycloheximide, extracts are capable of entering mitosis and repeatedly cycling between S phase and M phase, thus mimicking the cell cycle of the egg. This, together with the scope for biochemical manipulations, make Xenopus egg extracts ideal for studying the regulation of DNA replication and the role of nuclear structure in DNA replication.

4 Roles of Nuclear Structure in DNA Replication

The cell nucleus is the defining feature of eukaryotes. It is bounded by a nuclear envelope consisting of two concentric layers of membrane perforated by nuclear pores that serve as channels of communication between the nucleus and the cytoplasm. DNA is not randomly packed into the nucleus but packaged precisely in such a way that all regions are accessible for replication each cell cycle. Partial access and therefore partial replication would result in chromosome breakage or nondisjunction at mitosis, with disastrous consequences. The packing hierarchy involves radial loop organization from an axial scaffold, as well as the compaction resulting from coiling DNA twice around the nucleosome subunits of chromatin (Schedl and Grosveld 1995; Van Holde et al. 1995). A crucial feature of eukaryotic chromosomal DNA replication is that it always occurs within a nucleus. In lower eukaryotes such as fungi or Physarum, the nuclear membrane remains intact throughout the cell cycle, whereas it breaks down during mitosis of higher eukaryotes. Nevertheless, replication is constrained to interphase when the nuclear membrane is intact. We argue that this constraint has important regulatory consequences. Further key features of eukaryotic DNA replication are that multiple initiations occur within a single chromosome and that these initiations are coordinated so that each region of the chromosome replicates, but replicates only once in any cell cycle. We argue that nuclear structure has essential roles to play in coordinating multiple initiations to replicate the chromosome exactly once. NUCLEAR STRUCTURE IS REQUIRED FOR CELLULAR DNA REPLICATION Studies of eukaryotic...