Dirk Remus: Mechanism and Regulation of Origin Activation

Origin activation involves the unwinding of origin DNA by the replicative helicase and assembly of replisomes around the activated helicase. Ring-shaped hexameric helicases are widely used in all forms of life to unwind the parental DNA duplex during DNA replication. For example, in prokaryotes such as Escherichia coli, initiator proteins (DnaA, DnaC) load the hexameric helicase DnaB around single-stranded DNA at an origin site, upon which the helicase is active for both DNA unwinding and replication fork assembly. In various eukaryotic viruses, such as papilloma virus (E1 helicase) and simian virus (SV40, large T-antigen helicase), the helicase oligomerizes on its own to form double hexamers at the viral origin, upon which it induces origin melting and begins processive DNA unwinding, while simultaneously directing the formation of active replication forks from cellular components. In archaea, the replicative helicase is formed from a single Mcm subunit that assembles into single or double hexamers.

Strikingly, in eukaryotes, the replicative helicase comprises six distinct essential subunits (Mcm2-7), and helicase loading and activation are strictly consecutive events that require a complex cascade of accessory factors. While the tight regulation of helicase activation during origin firing allows the cell to coordinate DNA replication with other cell cycle processes such as chromosome segregation and cell division, it is not understood why so many additional factors are required to activate the eukaryotic helicase and to direct the formation of the replication forks, and why the helicase itself is a hetero-hexameric (or double-hexameric) assembly. Moreover, it is not known when and how the two strands of the parental DNA duplex are initially separated during origin activation and how Mcm2-7 generate and engage single-stranded DNA following loading around double-stranded DNA in the pre-RC.

Figure 6 Enlarge Image Figure 6 Budding yeast proteins involved in DNA replication. The indicated proteins assemble at chromosomal origin sites containing chromatin-bound Mcm2-7 complexes during origin activation in S phase and are involved in the formation replication forks. Boxes denote individual gene products; dashed circles indicate physical complexes; arrows indicate targets of the kinases that initiate origin-activation. Note: Many additional physical interactions exist between the complexes and individual proteins listed here, but are not indicated for clarity.

Higher-Order Protein Assembly

During origin activation in S phase chromatin-bound Mcm2-7 nucleate the assembly of replisomes at origin sites. The identification of the full complement of replisome components in eukaryotes is subject of ongoing research in the field. Figure 6 lists a number of the known budding yeast replication factors that bind to chromosomal sites containing Mcm2-7 during origin activation in S phase or that move together with Mcm2-7 in elongating replication forks. We aim to study how these different factors contribute to Mcm2-7 activation during origin firing and how their activities are coordinated at the replication fork. CDK promotes origin activation by directing the transient assembly of a complex of Sld3-Dpb11-Sld2 at replication origins. DDK promotes the activation of Mcm2-7 on chromatin by phosphorylating Mcm2-7 subunits directly. But how these events activate Mcm2-7 and lead to origin firing is not known. Do Sld3, Dpb11, and Sld2 simply serve as an assembly platform for other initiation factors, or do they perhaps also play more active roles during initiation, for example, by serving as allosteric activators in the pre-IC? If these proteins mediate the transition from the pre-RC to the pre-lC, which then are the respective factors they do interact with? Some proteins of the pre-lC, such as Cdc45 and GINS, remain associated with the actively unwinding Mcm2-7 helicase at the replication fork. How is the precise assembly of these accessory factors mediated and regulated during origin activation? Does their assembly require the exposure of binding surfaces in Mcm2-7 upon activation that are otherwise sequestered in the pre-RC? Or does the phosphorylation of Mcm2-7 generate novel binding epitopes? How do these accessory helicase subunits actually contribute to the Mcm2-7 DNA unwinding activity, and how do they contribute to the coordination of the Mcm2-7 helicase with the other replication fork activities, such as DNA polymerases?

Mechanism Of DNA Unwinding by Mcm2-7

Figure 7 Enlarge Image Figure 7 Possible DNA unwinding mechanisms for the Mcm2-7 helicase

Understanding the mechanism of DNA unwinding by Mcm2-7 will be critical for understanding the mechanisms that lead to Mcm2-7 activation. For example, it is not clear if the double hexamer is the active helicase form, or whether the component hexamers have to dissociate to be active. Although Mcm2-7 are loaded onto double-stranded DNA in the pre-RC, it is not clear whether in their active form they encircle single- or double-stranded DNA. Does DDK phosphorylation and/or accessory factor binding, therefore, for example, affect the structure of the DNA-bound Mcm2-7 double hexamer, perhaps by inducing the dissociation of the two hexamers, by inducing an ATP-hydrolysis-competent structural rearrangement, by exposing an Mcm2-7 domain that is required for strand separation during DNA unwinding, or perhaps by inducing the extrusion of one DNA strand from the hexamer channel? Reconstituting the activation of Mcm2-7 in vitro will allow the study of Mcm2-7 as a model for hexametric ring helicases in general. Conventional helicase assays involve the use of artificial substrates, such as short forked DNAs. Under such conditions both DNA translocases and DNA helicases may exhibit strand displacement activity. The use of a reconstituted loading system, which deposits the helicase on a double-stranded DNA substrate, may thus provide a more physiological experimental setup to study helicase mechanisms.

Identification of Replication Factors

Many of the initiation factors acting downstream of the pre-RC remain uncharacterized at the molecular level in vivo and their contribution to Mcm2-7 activation needs to be tested in vitro. But we may not know the full complement of activating factors yet. Moreover, various factors that are not essential for DNA replication are known to regulate and affect the activity of an origin in response to checkpoint signals or by modulating the chromatin state of the DNA template. Therefore, it will be important to characterize the full composition of the protein assemblies forming at replication origins during different stages of the cell cycle in vivo. Physical approaches are aimed at complementing existing genetic screens in the identification of both essential and non-essential replication factor.