Cell cycle regulation of DNA replication

Abstract

Eukaryotic DNA replication is regulated to ensure all chromosomes replicate once and only once per cell cycle. Replication begins at many origins scattered along each chromosome. Except for budding yeast, origins are not defined DNA sequences and probably are inherited by epigenetic mechanisms. Initiation at origins occurs throughout the S phase according to a temporal program that is important in regulating gene expression during development. Most replication proteins are conserved in evolution in eukaryotes and archaea, but not in bacteria. However, the mechanism of initiation is conserved and consists of origin recognition, assembly of prereplication (pre-RC) initiative complexes, helicase activation, and replisome loading. Cell cycle regulation by protein phosphorylation ensures that pre-RC assembly can only occur in G1 phase, whereas helicase activation and loading can only occur in S phase. Checkpoint regulation maintains high fidelity by stabilizing replication forks and preventing cell cycle progression during replication stress or damage.

Figure 1

Models of the regulation of DNA replication. (a) In coliphage λ replication, origin is recognized by λO protein, then λP protein loads hexameric DnaB helicase. λP protein is removed by DnaJ-K protein, which activates the helicase and allows replisome to replicate the DNA. (b) In eukaryotic DNA replication, origin is recognized by ORC, then Cdc6 and Cdt1 protein load the hexameric MCM helicase to form the “licensed” (L) pre-RC in G1 phase (L = 1, A = 0). Geminin inhibits Cdt1 and pre-RC formation. CDK and DDK become active in late G1, activate (A) the MCM helicase and load on the replisome that contains the DNA polymerases. In addition, CDK inhibits any further licensing (L = 0, A = 1). Toward this end, CDK phosphorylates Sld2 and Sld3 proteins and DDK phosphorylates MCM proteins, which “pushes out” the “A” domain of Mcm5.

Figure 2

Structures of ORC/Cdc6 and DNA helicases. (a) Ribbon diagram of the atomic structure of the N-terminal fragment of a single archaeal Mth-MCM subunit (b) rotated 90°. A, B, and C domains are indicated. Arrow indicates the position of the P62 residue (76). (c) EM reconstruction of yeast Orc/Cdc6 complex with ORC in blue (258) (d) EM reconstruction of the full-length archaeal double hexameric Mth-MCM complex (96). (e) Ribbon diagram of the atomic structure of a single hexamer of SV40 T antigen (161). (f) Space-filling diagram of the atomic structure of the N-terminal fragment of a single hexamer of the archaeal Mth-MCM complex (left) and a cut side-view (right) with two subunits removed for clarity; blue indicates positively charged amino acids, red indicates negatively charged amino acids (76).

Figure 3

Regulation of DNA replication by origin usage. (a) Prokaryotes have a single origin on a circular chromosome (above). (b) In eukaryotes, multiple origins are found on a single chromosome. When replication is “fast,” many origins are used, whereas only one origin is used in this region when replication is “slow”. Replication proceeds bidirectionally from an origin to form a replicon (below).

Figure 4

The clamp loading mechanism. Clamp loader (orange) opens clamp using the energy from ATP hydrolysis. Clamp loader is composed of “stator,” “wrench,” and “motor” functions. Clamp loader fixes clamp onto the “stator” while opening the clamp with the “wrench” and “motor.” Open clamp is bound to DNA and then closed. Adapted from (52).

Figure 5

Helicase substrates and models. (a) In vitro helicase substrates that are used frequently have small ssDNA (50 bp) annealed to ssDNA circle (5 kb) with nonhomologous 3′ tail. Helicases (red circle) such as SV40 T antigen or Mcm4/6/7 complex translocate 3′ to 5′ on the tail to unwind DNA and release oligonucleotide from the larger circle. Other substrates used resemble replication forks that are produced by annealing small ssDNA oligonucleotides with nonhomologous ends. Helicases can translocate 3′ to 5′ as above or 5′ to 3′ (DnaB). (b) A single hexameric helicase is depicted as a ring (blue) at the ends of a conventionally drawn replication fork. Lagging strand Okazaki fragments are shown with RNA (red) primers at their 5′ ends. (i) The SV40 T antigen model (161) is made by putting the two rings together forming a loop. In this model, the DNA is pumped into the channel of the double hexamer and then extruded out the holes in the outside C-terminal domains (Figure 2d). (ii) In the “pump-in-ring” model, each single hexamer translocates on a different strand of DNA (127). (iii) In the “ploughshare” model, the ploughshare (red) acts as a wedge and keeps the ssDNA unwound as it emerges from behind each single hexamer (271). (iv) In the “rotary pump” model, different single hexamers twist the DNA at a distance resulting in topological strain and unwinding in the center (151).

Figure 6

DNA replication and damage checkpoint regulation. Replication stress or blockage or DNA damage induces activation of a signal transduction pathway of many different proteins. The proteins are in different classes indicated as sensors, mediators, transducers, and effector targets. For example, if DNA replication is blocked, ssDNA coated by RPA sends a signal to activate Mec1 protein kinase. Mec1 binds to Mrc1, which amplifies the signal by binding to and activating Chk2 (Rad53) protein kinase. Chk2, in turn, inhibits Cdc45 helicase activation and loading of the replisome.