Daqi Yu

Background: Seasonal influenza A(H3N2) exhibits rapid global lineage turnover driven by continuous antigenic evolution. However, the emergence of newly dominant H3N2 lineages remains incompletely understood.Methods: We combined large-scale genomic surveillance with phylogenetic reconstruction, temporal mutation dynamics analyses, geographic dissemination analyses, and structural characterization to investigate emergence and global expansion of the H3N2 K lineage. A total of 170,793 complete human H3N2 hemagglutinin (HA) sequences collected globally between 1992 and 2026 were retrieved from GISAID.Results: We found that the K lineage rapidly replaced previously co-circulating H3N2 J.2 sublineages within a single influenza season. Its global expansion was associated with a non-canonical dissemination pattern centered on the United States rather than the traditional East–Southeast Asia (E-SEA) source system. Temporal mutation dynamics analyses of global surveillance data showed that K-lineage-defining substitutions were first detected in the United States and Japan. Despite accumulation of multiple antigenic-site substitutions, structural analyses suggested preservation of overall HA structural stability, with differential compatibility across pre-existing antibodies rather than complete antibody escape.Conclusions: The rapid rise of subclade K illustrates that contemporary H3N2 evolutionary centers are more geographically distributed than previously recognized. Our findings suggest that globally dominant H3N2 variants may emerge through reassembly of previously adaptive antigenic substitutions outside traditional E-SEA source regions. This study highlights the increasing complexity of global H3N2 evolutionary dynamics and underscores the importance of geographically balanced genomic surveillance for anticipating future antigenic and evolutionary trends.

Eukaryotic DNA replication requires the precise assembly of MCM2-7 single hexamers (SHs) into head-to-head double hexamers (DHs) at replication origins. While DH formation is well-characterized in budding yeast, the underlying mechanisms in human cells remain poorly understood. Here, we report cryo-electron microscopy structures of endogenous human MCM2-7 SH isolated from G1-phase cells. In these structures, human MCM2-7 adopts a latched spiral conformation in an autoinhibited state where the carboxyl-terminal extension (CTE) of MCM5 occupies the central channel, and MCM3-CTE is capable of locking the MCM2-5 gate to occlude DNA entry. Systematic functional analysis demonstrates that the six CTEs of MCM2-7 play distinct roles in SH stability, MCM loading, and DH formation on chromatin. Surprisingly, unlike in yeast, the human MCM3-CTE is dispensable for cell viability but ensures efficient genome-wide replication initiation. Our findings establish how human MCM2-7 enables flexible yet precise MCM loading via its CTEs, providing a framework for understanding the regulation of DNA replication initiation in higher eukaryotes.

In eukaryotes, the origin recognition complex (ORC) promotes the assembly of minichromosome maintenance 2 to 7 complexes into a head-to-head double hexamer at origin DNA in a process known as replication licensing. In this study, we present a series of cryoelectron microscopy structures of yeast ORC mutants in complex with origin DNA. We show that Orc6, the smallest subunit of ORC, utilizes its transcription factor II B-B domain to orchestrate the sequential binding of ORC to origin DNA. In addition, Orc6 plays the role of a scaffold by stabilizing the basic patch (BP) of Orc5 for ORC to capture and bend origin DNA. Importantly, disrupting DNA bending through mutating three key residues in Orc5-BP impairs ORC’s ability to promote replication initiation at two points during the pre-RC assembly process. This study dissects the multifaceted role of Orc6 in orchestrating ORC’s activities on DNA and underscores the vital role of DNA bending by ORC in replication licensing.

Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo–electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.

The circadian clock of cyanobacteria is based on an approximately 24h rhythm in the phosphorylation level of KaiC, a hexameric ATPase. This oscillation can be reconstituted in vitro by incubating three proteins together with ATP. Like all chemical oscillators, this system must include a nonlinear, or switch-like, feedback loop, whose nature has been unclear. Here, by using single particle cryo-EM at near-atomic resolution we identified two major conformational states of KaiC subunits, denoted as the exposed state and the buried state, which may provide a structural basis of how the KaiC hexamer changes its conformation during the (day-night) phosphorylation-dephosphorylation cycle. We classify the abundance and pattern of exposed and buried states within hexamers for more than 160,000 KaiC particles. The statistics of the spatial arrangement of the two states in hexamers can be quantitatively fit by a simple statistical physics model with an interaction energy between neighboring subunits and a local field that depends on phosphorylation state. Our study shows that phosphorylation shifts the probability of each conformation and reveals that there is substantial cooperativity between neighboring subunits, which can allow a KaiC hexamer to respond in an ultrasensitive, switch-like manner to changes in the phosphorylation level.