bims-replis 2025-06-08 papers

Nat Commun. 2025 Jun 05. 16(1): 5230

An archaeal nucleoid-associated protein binds an essential motif in DNA replication origins.

Rajkumar Dhanaraju, Rachel Y Samson, Xu Feng, Alessandro Costa, Giovanni Gonzalez-Gutierrez, Stephen D Bell.

DNA replication typically has defined start sites, or replication origins, which are designated by their recognition by specific initiator proteins. In addition to initiators, general chromatin or nucleoid-associated proteins have been shown to play roles in modulating origin efficiency in eukaryotes and bacteria. The role of chromatin proteins in origin function in the archaeal domain of life is poorly understood. Here, we describe a dissection of sequences elements required for in vivo function of an archaeal DNA replication origin. Our data reveal a hitherto uncharacterized sequence element, the ucm, is required for origin activity. We identify a protein, UBP, that interacts with the ucm and additionally with hundreds of other sites on the genome. We solve the crystal structure of UBP alone and in complex with ucm DNA, and further show that UBP interacts with the MCM replicative helicase. Taken together, our data provide evidence that UBP functions as a general nucleoid-associated protein that plays a key role in facilitating the egress of the MCM replicative helicase from DNA replication origins.

DOI: https://doi.org/10.1038/s41467-025-60618-3

bioRxiv. 2025 May 13. pii: 2025.05.09.652650. [Epub ahead of print]

An Orc6 tether mediates ORC binding site switching during replication origin licensing.

David Driscoll, Larry J Friedman, Jeff Gelles, Stephen P Bell.

During origin licensing, the origin recognition complex (ORC) loads two Mcm2-7 helicases onto DNA in a head-to-head conformation, establishing the foundation for subsequent bidirectional replication. Single-molecule experiments support a helicase-loading model in which one ORC loads both Mcm2-7 helicases at origins. For this to occur, ORC must release from its initial Mcm2-7 and DNA binding sites, flip over the helicase, and bind the opposite end of the Mcm2-7 complex and adjacent DNA to form the MO complex. Importantly, this binding-site transition occurs without ORC releasing into solution. Using a single-molecule FRET assay, we show that the N-terminal half of Orc6 tethers ORC to the N-terminal tier of Mcm2-7 (Mcm2-7N) during ORC's binding-site transition. This interaction involves both the folded Orc6 N-terminal domain (Orc6N) and the adjacent unstructured linker and forms before ORC releases from its initial Mcm2-7 interaction. The absence of this interaction increases the rate of ORC release into solution, consistent with a tethering function. CDK phosphorylation of ORC inhibits the tethering interaction, providing a mechanism for the known CDK inhibition of MO complex formation. Interestingly, we identify mutations in the Orc6 linker region that support MO complex formation but prevent double-hexamer formation by inhibiting stable second Mcm2-7 recruitment. Our study provides a molecular explanation for a one-ORC mechanism of helicase loading and demonstrates that Orc6 is involved in multiple stages of origin licensing.
Significance Statement: Bidirectional DNA replication is critical for accurate and complete duplication of the genome. Eukaryotic organisms coordinate this through loading of two oppositely-oriented Mcm2-7 replicative helicases at origins of replication. Using single-molecule biochemical studies, we identified and characterized a tethering interaction during helicase loading that enables the helicase loader ORC (origin recognition complex) to flip between two Mcm2-7 and DNA binding sites to load the second helicases in the opposite orientation. This interaction is cell-cycle regulated as part of the mechanisms ensuring replication from a given origin initiates only once. Our findings have important implications for the multiple mechanisms of helicase loading and illustrate how single-molecule studies can complement structural studies to provide a full view of complex molecular assembly events.

DOI: https://doi.org/10.1101/2025.05.09.652650

Nat Commun. 2025 Jun 04. 16(1): 5176

Assembly and breakage of head-to-head double hexamer reveals mpox virus E5-catalyzed DNA unwinding initiation.

Yingxian Cheng, Pu Han, Qi Peng, Ruili Liu, Hao Liu, Bin Yuan, Yunfei Zhao, Lu Kuai, Jianxun Qi, Kai Miao, Yi Shi, George Fu Gao, Han Wang.

The replicative helicase-catalyzed unwinding of the DNA double helix is the initiation of DNA replication. Helicases and primases are functionally related enzymes that have even been expressed as fusion proteins in some organisms and viruses. However, the mechanism underlying DNA unwinding initiation by these helicase-primase fusion enzymes and the functional association between domains have not been elucidated. Herein, we report the cryo-EM structures of mpox virus E5, the founding member of these helicase-primase enzymes, in various enzymatic stages. Notably, E5 forms a head-to-head double hexamer encircling dsDNA, disrupted by the conformational rearrangement of primase domains upon nucleotide incorporation. Five E5-ssDNA-ATP structures further support an ATP cycle-driven non-classical escort model for E5 translocation. Finally, the helicase domain is found to enhance the primase function as a DNA scaffold. Together, our data shed light on the E5-mediated DNA unwinding model including dsDNA loading, DNA melting, ssDNA translocation, and provide a reasonable interpretation for evolutionary preservation of helicase-primase fusion from a functional perspective.

DOI: https://doi.org/10.1038/s41467-025-60539-1