The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5′-3′ through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol e below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle doublestranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.CMG helicase | DNA replication | DNA polymerase | origin initiation | replisome R eplicative helicases are hexameric rings in all domains of life (1-3). In bacteria and archaea, the replicative helicase is a homohexamer and encircles single-strand (ss) DNA at a replication fork. Some viral and phage replicative helicases are also ring-shaped hexamers, including bovine papilloma virus (BPV) E1, simian virus 40 (SV40) large T-antigen (T-Ag), and the T4 and T7 phage helicases. Unlike other replicative helicases, the eukaryotic replicative Mcm2-7 helicase is composed of six nonidentical but homologous Mcm subunits that become activated upon assembly with five accessory factors (Cdc45 and GINS tetramer) to form the 11-subunit CMG (Cdc45, Mcm2-7, GINS) (4-6). Numerous studies have outlined the process that forms CMG at origins in which the Mcm2-7 heterohexamer is loaded onto DNA as an inactive double hexamer in G1 phase, and becomes activated in S phase by several initiation proteins and cell-cycle kinases that assemble Cdc45 and GINS onto Mcm2-7 to form the active CMG helicases (7-9).Helicases assort into six superfamilies (SF1-SF6) based on sequence alignments (10). The SF1 and SF2 helicases are generally monomeric and the SF3-SF6 helicases are hexameric rings used in DNA replication and other processes. The bacterial SF4 and SF5 helicases contain RecA-based motors and translocate 5′-3′, whereas the eukarytic SF3 and SF6 helicases contain AAA+ (ATPases associated with diverse cellular activities)-based motors and translocate 3′-5′ (3, 10). Examples of well-studied hexameric helicases include t...

The current view is that eukaryotic replisomes are independent. Here we show that Ctf4 tightly dimerizes CMG helicase, with an extensive interface involving Psf2, Cdc45, and Sld5. Interestingly, Ctf4 binds only one Pol α-primase. Thus, Ctf4 may have evolved as a trimer to organize two helicases and one Pol α-primase into a replication factory. In the 2CMG–Ctf43–1Pol α-primase factory model, the two CMGs nearly face each other, placing the two lagging strands toward the center and two leading strands out the sides. The single Pol α-primase is centrally located and may prime both sister replisomes. The Ctf4-coupled-sister replisome model is consistent with cellular microscopy studies revealing two sister forks of an origin remain attached and are pushed forward from a protein platform. The replication factory model may facilitate parental nucleosome transfer during replication.

Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome β5i subunit while sparing the constitutive β5c subunit. Here we report β5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively, an AsnEDA inhibitor binds the active site. Hydrophobic interactions are accompanied by hydrogen bonding with β5i and β6 subunits. The inhibitors are far more cytotoxic for myeloma and lymphoma cell lines than for hepatocarcinoma or non-activated lymphocytes. They block human B-cell proliferation and promote apoptotic cell death selectively in antibody-secreting B cells, and to a lesser extent in activated human T cells. Reversible, β5i-selective inhibitors may be useful for treatment of diseases involving activated or neoplastic B cells or activated T cells.

The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.

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In eukaryotes, DNA replisome is a large protein complex that carries out DNA replication, which contains several enzymatic protein subunits, such as helicase, RNA primase and DNA polymerase. Replisome generates a DNA replication fork and duplicates both the leading and lagging strand of duplex DNA. Previous studies showed the Ctf4 trimer is proposed to be a hub to coordinate the subunits in replisome, Ctf4 trimer may link CMG and Pol a through binding CIP motif of Sld5 and Pol a. By using single‐particle cryo‐EM of an active CMG on the forked DNA with a dual biotin‐streptavidin block, we solved the first structure of helicase in complex with a DNA fork. The DNA duplex stem penetrates into the central channel of the NTD tier and the unwound leading ssDNA traverses the channel through the NTD tier into the CTD motor tier, 5′‐3′ through CMG. Therefore, during translocation the CTD motor is behind the NTD ring that is pushed ahead to face the fork, opposite the currently accepted polarity. The polarity of the NTD ahead of the CTD places the leading Polɛ below CMG and Polα‐primase at the top of CMG, above the unwinding point of the replication fork. The DNA direction requires that the Mcm rings must first pass one another when the Mcm2‐7 double hexamer come apart. Interestingly, the new NTD‐to‐CTD polarity of translocation reveals an unforeseen quality control mechanism that each Mcm2‐7 is fully converted from encircling dsDNA to ssDNA before they can depart from the origin. We propose this architecture of two motors head to head may generate energy that underlies initial melting at the origin.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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