Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

The human pre-replication complex is an open complex.

In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.

The Origin Recognition Complex: From Origin Selection to Replication Licensing in Yeast and Humans.

Understanding human DNA replication through the study of yeast has been an extremely fruitful journey. The minichromosome maintenance (MCM) 2-7 genes that encode the catalytic core of the eukaryotic replisome were initially identified through forward yeast genetics. The origin recognition complexes (ORC) that load the MCM hexamers at replication origins were purified from yeast extracts. We have reached an age where high-resolution cryoEM structures of yeast and human replication complexes can be compared side-by-side. Their similarities and differences are converging as alternative strategies that may deviate in detail but are shared by both species.

Structural Insight into the MCM double hexamer activation by Dbf4-Cdc7 kinase.

The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation by phosphorylation of the MCM double hexamer (MCM-DH) to promote helicase activation. Here, we determine a series of cryo electron microscopy (cryo-EM) structures of yeast DDK bound to the MCM-DH. These structures, occupied by one or two DDKs, differ primarily in the conformations of the kinase core. The interactions of DDK with the MCM-DH are mediated exclusively by subunit Dbf4 straddling across the hexamer interface on the three N-terminal domains (NTDs) of subunits Mcm2, Mcm6, and Mcm4. This arrangement brings Cdc7 close to its only essential substrate, the N-terminal serine/threonine-rich domain (NSD) of Mcm4. Dbf4 further displaces the NSD from its binding site on Mcm4-NTD, facilitating an immediate targeting of this motif by Cdc7. Moreover, the active center of Cdc7 is occupied by a unique Dbf4 inhibitory loop, which is disengaged when the kinase core assumes wobbling conformations. This study elucidates the versatility of Dbf4 in regulating the ordered multisite phosphorylation of the MCM-DH by Cdc7 kinase during helicase activation.

Humanizing the yeast origin recognition complex.

The Origin Recognition Complex (ORC) is an evolutionarily conserved six-subunit protein complex that binds specific sites at many locations to coordinately replicate the entire eukaryote genome. Though highly conserved in structure, ORC's selectivity for replication origins has diverged tremendously between yeasts and humans to adapt to vastly different life cycles. In this work, we demonstrate that the selectivity determinant of ORC for DNA binding lies in a 19-amino acid insertion helix in the Orc4 subunit, which is present in yeast but absent in human. Removal of this motif from Orc4 transforms the yeast ORC, which selects origins based on base-specific binding at defined locations, into one whose selectivity is dictated by chromatin landscape and afforded with plasticity, as reported for human. Notably, the altered yeast ORC has acquired an affinity for regions near transcriptional start sites (TSSs), which the human ORC also favors.

Structure of the origin recognition complex bound to DNA replication origin.

The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3 Å cryo-electron microscopy structure of the Saccharomyces cerevisiae ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.

Unique Roles of the Non-identical MCM Subunits in DNA Replication Licensing.

A family of six homologous subunits, Mcm2, -3, -4, -5, -6, and -7, each with its own unique features, forms the catalytic core of the eukaryotic replicative helicase. The necessity of six similar but non-identical subunits has been a mystery since its initial discovery. Recent cryo-EM structures of the Mcm2-7 (MCM) double hexamer, its precursors, and the origin recognition complex (ORC)-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate showed that each of these subunits plays a distinct role in orchestrating the assembly of the pre-replication complex (pre-RC) by ORC-Cdc6 and Cdt1.

Open-ringed structure of the Cdt1-Mcm2-7 complex as a precursor of the MCM double hexamer.

The minichromosome maintenance complex (MCM) hexameric complex (Mcm2-7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1-Mcm2-7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1-MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10-15-Å gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.

Structure of the MCM2-7 Double Hexamer and Its Implications for the Mechanistic Functions of the Mcm2-7 Complex.

The eukaryotic minichromosome maintenance 2-7 complex is the core of the inactive MCM replication licensing complex and the catalytic core of the Cdc45-MCM-GINS replicative helicase. The years of effort to determine the structure of parts or the whole of the heterohexameric complex by X-ray crystallography and conventional cryo-EM produced limited success. Modern cryo-EM technology ushered in a new era of structural biology that allowed the determination of the structure of the inactive double hexamer at an unprecedented resolution of 3.8 Å. This review will focus on the fine details observed in the Mcm2-7 double hexameric complex and their implications for the function of the Mcm2-7 hexamer in its different roles during DNA replication.

Structure of the eukaryotic MCM complex at 3.8 Å.

DNA replication in eukaryotes is strictly regulated by several mechanisms. A central step in this replication is the assembly of the heterohexameric minichromosome maintenance (MCM2-7) helicase complex at replication origins during G1 phase as an inactive double hexamer. Here, using cryo-electron microscopy, we report a near-atomic structure of the MCM2-7 double hexamer purified from yeast G1 chromatin. Our structure shows that two single hexamers, arranged in a tilted and twisted fashion through interdigitated amino-terminal domain interactions, form a kinked central channel. Four constricted rings consisting of conserved interior ß-hairpins from the two single hexamers create a narrow passageway that tightly fits duplex DNA. This narrow passageway, reinforced by the offset of the two single hexamers at the double hexamer interface, is flanked by two pairs of gate-forming subunits, MCM2 and MCM5. These unusual features of the twisted and tilted single hexamers suggest a concerted mechanism for the melting of origin DNA that requires structural deformation of the intervening DNA.

Novel features of ARS selection in budding yeast Lachancea kluyveri.

BACKGROUND: The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined. RESULTS: In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis. CONCLUSIONS: Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

A comprehensive genome-wide map of autonomously replicating sequences in a naive genome.

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins. The machinery that initiates DNA synthesis is highly conserved, but the sites where the replication initiation proteins bind have diverged significantly. Functional comparative genomics is an obvious approach to study the evolution of replication origins. However, to date, the Saccharomyces cerevisiae replication origin map is the only genome map available. Using an iterative approach that combines computational prediction and functional validation, we have generated a high-resolution genome-wide map of DNA replication origins in Kluyveromyces lactis. Unlike other yeasts or metazoans, K. lactis autonomously replicating sequences (KlARSs) contain a 50 bp consensus motif suggestive of a dimeric structure. This motif is necessary and largely sufficient for initiation and was used to dependably identify 145 of the up to 156 non-repetitive intergenic ARSs projected for the K. lactis genome. Though similar in genome sizes, K. lactis has half as many ARSs as its distant relative S. cerevisiae. Comparative genomic analysis shows that ARSs in K. lactis and S. cerevisiae preferentially localize to non-syntenic intergenic regions, linking ARSs with loci of accelerated evolutionary change.

Novel DNA binding properties of the Mcm10 protein from Saccharomyces cerevisiae.

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a approximately 1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21-24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20-50 nucleotides was approximately 3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.