MCM2-7 proteins provide essential helicase functions in eukaryotes at chromosomal DNA replication forks. During the G1 phase of the cell cycle, they remain loaded on DNA but are inactive. We have used recombinant methods to show that the Drosophila MCM2-7 helicase is activated in complex with Cdc45 and the four GINS proteins (CMG complex). Biochemical activities of the MCM AAA+ motor are altered and enhanced through such associations: ATP hydrolysis rates are elevated by two orders of magnitude, helicase activity is robust on circular templates, and affinity for DNA substrates is improved. The GINS proteins contribute to DNA substrate affinity and bind specifically to the MCM4 subunit. All pairwise associations among GINS, MCMs, and Cdc45 were detected, but tight association takes place only in the CMG. The onset of DNA replication and unwinding may thus occur through allosteric changes in MCM2-7 affected by the association of these ancillary factors.

Two central steps for initiating eukaryotic DNA replication involve loading of the Mcm2–7 helicase onto double-stranded DNA and its activation by GINS–Cdc45. To better understand these events, we determined the structures of Mcm2–7 and the CMG complex by using single-particle electron microscopy. Mcm2–7 adopts two conformations—a lock-washer-shaped spiral state and a planar, gapped-ring form—in which Mcm2 and Mcm5 flank a breach in the helicase perimeter. GINS and Cdc45 bridge this gap, forming a topologically closed assembly with a large interior channel; nucleotide binding further seals off the discontinuity between Mcm2 and Mcm5, partitioning the channel into two smaller pores. Together, our data help explain how GINS and Cdc45 activate Mcm2–7, indicate that Mcm2–7 loading may be assisted by a natural predisposition of the hexamer to form open rings, and suggest a mechanism by which the CMG complex assists DNA strand separation.

Summary Eukaryotic cells license far more origins than are actually used for DNA replication, thereby generating a large number of dormant origins. Accumulating evidence suggests that such origins play a role in chromosome stability and tumor suppression, though the underlying mechanism is largely unknown. Here, we show that a loss of dormant origins results in an increased number of stalled replication forks even in unchallenged S phase in primary mouse fibroblasts derived from embryos homozygous for the Mcm4Chaos3 allele. We found that this allele reduces the stability of the MCM2-7 complex, but confers normal helicase activity in vitro. Despite the activation of multiple fork recovery pathways, replication intermediates in these cells persist into M phase, increasing the number of abnormal anaphase cells with lagging chromosomes and/or acentric fragments. These findings suggest that dormant origins constitute a major pathway for stalled fork recovery, contributing to faithful chromosome segregation and tumor suppression.

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.DOI: http://dx.doi.org/10.7554/eLife.03273.001

Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs.Papillomavirus (PV) E2 protein is a central regulator of the viral life cycle. In addition to its well-established activity as a transcription modulator and replication initiator protein (6), E2 of bovine papillomavirus type 1 (BPV1) has recently emerged as a trans factor which mediates mitotic segregation of viral genomes by tethering them to host cell chromatin (7,12,19). The first candidate for a receptor of E2 in the latter process, Brd4, is attached to the chromatin through its two bromodomains, which bind to acetylated histones H3 and H4 both in interphase and in mitosis (4, 25). Mutated E2 proteins that are defective in Brd4 binding are unable to bind to mitotic chromosomes (2), and ectopic expression of Brd4 can reconstitute the BPV1 E2-dependent extrachromosomal plasmid maintenance in the yeast Saccharomyces cerevisiae, where such a process normally does not function (3). Ectopic expression of the E2-binding C-terminal domain (CTD) of Brd4 in mammalian cells disrupts the interaction of E2 with cellular Brd4 and relocates E2 from mitotic chromosomes (25,26). Brd4 CTD binds to the N-terminal domain of E2 (25), which is also responsible for interactions critical for transcription activation and replication initiator activities of E2. Therefore, we suspected that Brd4 might have a more complex role in the PV life cycle than initially proposed. We tested this idea in the present study and show that the Brd4 bromodomain protein can indeed participate in the BPV1 E2-dependent transcription activation and DNA replication processes. Brd4 is specifically involved in the E2-activated transcription process; the role of Brd4 in BPV1 DNA replication, however, is either largely or completely independent of its binding to E2. Our data demonstrate the possible involvement of Brd4 also in polyomavirus DNA replication and reveal the varying importance of the Brd4-linked component for BPV1 DNA replication in different cell lines.Cloning of the dominant-negative form of Brd4 (Brd4 CTD). The use of a dominant-negative truncated version of Brd4 is a useful alternative to manipulations with a full-length gene, as overexpression or knockout of Brd4 in mammalian cells has been shown to cause severe alterations in cell growth (5, 15). Overexpression of Brd4 CTD affects neither the growth of several cell lines (C127, C33A, HeLa) (25, 26) nor the cell cycle dist...