While the principal DNA replication machinery is conserved among eukaryotes, budding yeast offers several practical advantages for the study of DNA replication, such as ease of genetic manipulation, efficient methods for conditional cell cycle synchronization, as well as relatively short (100-150 bp) and well-defined replication origins, the chromosomal start sites for DNA replication.
DNA replication initiation is a complex, multistep reaction that involves the ordered assembly and concerted transient activities of a series of proteins and protein complexes at chromosomal origin sites. The product of these origin-associated events is a pair of “replisomes,” large molecular machines that contain the replication fork and all associated activities required for DNA synthesis along the chromosomal template. Although many essential eukaryotic initiation factors, and their sequential order of origin association, have been identified, molecular functions for these factors often remain undefined. Understanding eukaryotic DNA replication initiation will therefore require the identification of the full complement of polypeptides present in the complexes involved in initiation, characterization of their structures and biochemical activities, and mapping of the protein-protein and protein-DNA contacts occurring at replication origins during initiation. To this end we employ a combination of genetic and biochemical approaches to identify and characterize the protein complexes acting at replication origins and replication forks in budding yeast in vivo, and test their ability in purified form to reconstitute the various steps involved in initiation in vitro.
A Two-Step Strategy for Initiating DNA Replication in Eukaryotes
Two-step model for the initiation of DNA replication in eukaryotes
In eukaryotes, DNA replication initiates from a large number of origin sites distributed on multiple chromosomes. Origin firing, therefore, needs to be carefully coordinated and regulated to ensure, on the one hand, that a sufficient number of origins per chromosome fires in any given cell cycle to achieve complete replication of the entire genome, and to prevent, on the other hand, local re-replication due to reinitiation from the same origin site during one cell cycle. This tight regulation is achieved by a conserved two-step mechanism (Figure 1). In the first step, termed origin licensing, the hexameric replicative helicase, Mcm2-7, is loaded in an inactive form into pre-replicative complexes (pre-RCs) at potential origin sites. This helicase loading step requires the ATP-dependent activity of the Origin Recognition Complex (ORC), which marks potential origin sites on the chromosome, Cdc6, which like several of the ORC subunits is a member of the AAA+ family of ATPases, as well as Cdt1, a loading factor that in budding yeast forms a complex with Mcm2-7 prior to loading. Pre-RCs can only assemble at the end of mitosis and during G1 phase, when cyclin-dependent kinase (CDK) activity is low and the anaphase promoting complex/cyclosome (APC/C) is active, because CDKs inhibit the activities of each pre-RC component.
Purified pre-RC proteins
We have recently reconstituted the Mcm2-7 loading reaction with purified budding yeast proteins (Figure 2) and found that ORC and Cdc6 cooperatively load two heptamers of the Cdt1·Mcm2-7 complex into a head-to-head Mcm2-7 double hexamer onto DNA (Figure 3). This suggests a molecular mechanism for the establishment of bi-directional DNA replication forks. Unexpectedly, Mcm2-7 double hexamers are mobile and can passively slide on the DNA, which is consistent with the electron-microscopic observation of DNA passing through the Mcm2-7 double hexamer (Figure 4), and which may suggest that Mcm2-7 is loaded around double-stranded DNA. The formation of a double hexameric Mcm2-7 complex around DNA has important implications for the DNA unwinding mechanism by the Mcm2-7 helicase, which is currently unknown. Consistent with the two-step mechanism for origin activation, MCM2-7 is inactive following loading onto DNA in vitro.
Three-dimensional EM reconstruction of budding yeast Mcm2-7 double hexamer. Top: Model; Middle: Surface representations; Bottom: Cross-section to highlight central channel. N =N-terminal domain, AAA+ = AAA+ ATP-binding domain.
Chromosomal sites containing functional pre-RCs serve as binding sites for additional replication factors including Mcm10, Sld3, and Cdc45. The molecular functions of these proteins are unclear, but they are required for origin activation in the second step, which occurs throughout S phase. Origin activation is induced by the activities of two protein kinases, CDK and Cdc7·Dbf4 (DDK, for Dbf4-dependent kinase).
These kinases promote the activation of a subset of “licensed” origins by activating the MCM2-7 helicase and by promoting the assembly of further initiation factors around pre-RCs to form pre-initiation complexes (pre-IC) that are ultimately converted into replisomes (Figure 1).
CDKs thus play a dual role in regulating DNA replication initiation that is central to the prevention of re-replication: They inhibit the formation of new pre-RCs by phosphorylating ORC, Cdc6, and free Cdt1, Mcm2-7, while activating existing pre-RCs via phosphorylation of the down-stream factors Sld2 and Sld3, which promotes their physical interaction with the tandem BRCT repeat-containing protein Dpb11.
Mcm2-7 double hexamer bound to circular 1 kb origin-containing DNA visualized by transmission electron microscopy after tungsten rotary shadow casting.
Formation of the Sld3-Dpb11-Sld2 complex is only transiently required for origin activation as these factors are not present at elongating replication forks. Yet, how they actually promote origin activation is unknown. Cdc7·Dbf4 phosphorylation promotes origin firing by directly phosphorylating chromatin-bound Mcm2-7 complexes, which presumably activates the helicase activity of Mcm2-7. How this activation is achieved is not clear, but Cdc7·Dbf4-dependent phosphorylation might induce conformational changes in Mcm2-7 that de-inhibit the helicase activity, and/or might promote the association of accessory helicase factors with Mcm2-7 such as Cdc45 and the tetrameric GINS complex.
A major challenge will therefore be to define the determinants of Mcm2-7 activation in vitro, which will give insight into the mechanisms that regulate the Mcm2-7 helicase, into the structure and composition of the active form of the Mcm2-7 helicase, and into the mechanism of DNA unwinding by Mcm2-7. This information is critical for understanding the architecture of the eukaryotic replisome, at the center of which acts the Mcm2-7 helicase.