The Temporal Regulation of S Phase Proteins During G1

Abstract

Successful DNA replication requires intimate coordination with cell-cycle progression. Prior to DNA replication initiation in S phase, a series of essential preparatory events in G₁ phase ensures timely, complete, and precise genome duplication. Among the essential molecular processes are regulated transcriptional upregulation of genes that encode replication proteins, appropriate post-transcriptional control of replication factor abundance and activity, and assembly of DNA-loaded protein complexes to license replication origins. In this chapter we describe these critical G₁ events necessary for DNA replication and their regulation in the context of both cell-cycle entry and cell-cycle progression.

Keywords: APC/C; CDK; Cell cycle; Cyclin; DREAM complex; E2F; Origin licensing; Quiescence; RB.

Figure 1. Activities of cell cycle dependent kinases and phosphatases during G₁ and G₀

Darker shading indicates more activity. Dashed lines indicate entry and exit from G₀.

Figure 2. E2F and RB proteins

A. A diagram of the motifs of the transcription factor E2F1 as an example activator E2F. DBD, DNA binding domain; DP1, DP binding domain; RB, RB binding domain. B. RB phosphorylation sites. Blue indicates increased possible binding affinity to E2F transcription factors; red indicates decreased affinity and black indicates no change in affinity to E2F transcription factors. The dashed lines indicate where RB binds E2F1.

Figure 3. Activities of the pocket proteins RB, p107, and p130, and activating E2F transcription factors during G₁ and G₀

Darker shading indicates more activity. Dashed lines indicate entry and exit from G₀.

Figure 4. Inactivation of E2F target genes by the RB associated SAP30-mSin3-HDAC complex in G₁

Monophosphorylated RB binds activating E2F transcription factors at promoters of E2F target genes. The SAP30-mSin3-HDAC complex binds to RB and the associated HDACs deacetylate the E2F target gene, repressing transcription. Hyperphosphorylated RB releases E2F and dissociates from the SAP30-mSin3-HDAC complex, relieving the transcriptional repression of E2F target genes.

Figure 5. Inactivation of E2F by monophosphorylation of RB

In early G_1, E2F is bound and inactivated by mono-phosphorylated RB. Later in G₁, cyclin E/CDK2 becomes active and hyperphosphorylates RB, releasing E2F which then activates transcription of target genes.

Figure 6. Origin licensing

DNA replication origins are licensed by loading MCM complexes. The first CDT1 associated MCM complex is loaded onto DNA through the action of the DNA bound origin recognition complex (ORC) and CDC6. Once the first MCM complex is successfully loaded by ORC, CDC6 and CDT1, CDC6 and CDT1 dissociate from the DNA bound MCM and ORC complexes. A second CDC6 molecule then associates with the DNA bound complexes. Finally, a second CDT1 associated MCM complex is loaded onto the DNA in a head to head fashion and CDC6 and CDT1 are released.

Figure 7. Modifications and activation of APC/C^CDH1 from mitosis to the onset of S phase

In late mitosis, CDH1 replaces CDC20 as the APC/C E3 ligase and is active during early G₁. Two APC/C^CDH1 substrates in early G1 are CDC20 and the origin licensing protein, CDC6. In late G₁ Cyclin E/CDK2 phosphorylates and protects CDC6. Also in late G1, the pseudosubstrate, EMI1, binds to CDH1, inactivating the complex. In late G₁ and early S phase, CDH1 is phosphorylated by cyclin E/CDK2 and cyclin A/CDK2, respectively; this phosphorylation dissociates CDH1 from APC/C, thus inactivating the complex. From S phase through mitosis, the E3 ligase CDH1 is polyubiquitylated by the SCF complex and degraded by the 26S proteasome.