Preventing re-replication of chromosomal DNA

Abstract

To ensure its duplication, chromosomal DNA must be precisely duplicated in each cell cycle, with no sections left unreplicated, and no sections replicated more than once. Eukaryotic cells achieve this by dividing replication into two non-overlapping phases. During late mitosis and G1, replication origins are 'licensed' for replication by loading the minichromosome maintenance (Mcm) 2-7 proteins to form a pre-replicative complex. Mcm2-7 proteins are then essential for initiating and elongating replication forks during S phase. Recent data have provided biochemical and structural insight into the process of replication licensing and the mechanisms that regulate it during the cell cycle.

Figure 1

Regulated loading and unloading of Mcm2-7 during the cell cycle. A small segment of chromosomal DNA that encompasses 3 replication origins is shown. At the end of mitosis (M), the replication licensing system (RLS) is activated, which causes Mcm2-7 (the asymmetric rectangles marked ‘M’) to be loaded onto potential replication origins (origin licensing). The licensing system is turned off at the end of G1 by inhibition by CDKs and/or geminin. During S phase, the Mcm2-7 complexes are displaced from replicated DNA by moving ahead of the replication fork, and are removed from DNA at fork termination. In this way, replicated DNA cannot undergo further initiation events until passage through mitosis.

Figure 2

Crystal structure of the N terminus of the MtMCM. A - C. Different views of the N terminus of the MtMCM dodecamer, highlighting the central channel that runs through it. Positive charges are shaded in blue, negative charges in red. A, End view. B, Side view. C, Same view as B, but with the two front-most monomers removed to reveal the central channel. Yellow arrows show the side channels passing between exterior and interior. Reproduced from reference. D A side view of the electron density of full-length MtMCM, aligned to approximately correspond to the size view of the crystal structure shown in B. In green, crystal structure of the N terminus has been fitted into the electron density. In red is fitted a hexameric model of the core AAA+ domain of RuvB, which has significant homology to the C-terminus of the Mcm2-7 proteins. Reproduced from reference.

Figure 3

Model for SV40 T antigen function at replication initiation. Model for the function of the SV40 T antigen based on the crystal structure of the helicase domain. A, A double hexamer of the SV40 T antigen, comprising a bilobed helicase domain and an origin binding domain (obd), is shown encircling origin DNA. B, C, D, Model for the possible extrusion of unwound DNA through a side channel of the helicase domain to unwind the two forks bidirectionally. A, T antigen binds and encircles origin DNA. B, Origin DNA is unwound, possibly by the two T antigen hexamers rotating relative to one another. C, A conformational change to form the fully initiated complex may involve extrusion of one of the single strands through an exit channels in the helicase domain, with the other single strand being extruded at the hexamer-hexamer interface. Reproduced from reference.

Figure 4

Stepwise assembly of pre-replicative complex (pre-RC) proteins during origin licensing. A, ORC is first recruited to the replication origin. B, ORC recruits Cdc6 and Cdt1. C, ORC, Cdc6 and Cdt1 act together to load multiple Mcm2-7 hexamers onto the origin, which licenses the DNA for replication. D, Initiation-competent complexes are probably formed by the back-to-back assembly of two Mcm2-7 complexes. Since ORC is asymmetrical, this might require a second ORC molecule in the opposite orientation to load Mcm2-7 in the opposite orientation.

Figure 5

Cell cycle regulation of the licensing system in yeasts and metazoans. The activity of components of the licensing system during the cell cycle of yeasts (A) and metazoans (B) is shown. In the lower part of each figure, the licensing system is shown active (green) only in G1. In yeasts, licensing is inhibited at other times by CDKs (blue), whilst in metazoans, licensing is inhibited in S phase and G2 by geminin (orange) and in mitosis by a combination of geminin and CDKs (purple). Above this, the activity of different pre-RC components (ORC, Cdc6, Cdt1 and Mcm2-7) is shown: green for active, red for inhibited.

Figure 6

Structure of a geminin : Cdt1 complex. Structure of a complex between the central regions of geminin (tGeminin, in green) and Cdt1 (tCdt1, in orange). The N-terminal ten residues are structured in the tGeminin monomer that binds to tCdt1 but are disordered in the other tGeminin monomer, indicating that this region might undergo induced folding after binding to tCdt1. Comparison with the structure of geminin on its own suggests that the kink at the C terminal end of geminin may be a distortion due to a packing interaction. Reproduced from reference.

Figure 7

The possible role of cyclin E in promoting re-licensing of DNA on exit from G0. Mcm2-7 (red shapes ‘M’) are shown on a small segment of chromosomal DNA in a cell either passing directly through G1 (top arrow) or passing into G0 and then into S phase (lower arrows). During passage through G1, Mcm2-7 loaded onto DNA in late mitosis remains stably bound to DNA. Geminin is activated in late G1, just prior to entry into S phase. From G1, cells also have the option of entering quiescence (the G0 state). On entry into G0 from G1, the DNA is de-licensed and Mcm2-7, Cdc6 and Cdt1 are degraded. This might provide a barrier that prevents the inappropriate proliferation of these cells. On exit from G0 into S phase, quiescent cells must relicense their DNA, and cyclin E is required for this re-licensing. Cyclin E might be required to drive the re-synthesis or activation of Mcm2-7, Cdc6 or Cdt1, or cyclin E might be required to temporarily suppress geminin activity, which is also resynthesized prior to entry into S phase.