Biology subjects

Eickhoff, P.

Publications and source records attributed to Eickhoff, P..

3 recordsLinked to original sources

bioRxiv2024

Chromosome end protection by RAP1-mediated inhibition of DNA-PK

During classical non-homologous end joining (cNHEJ), DNA-dependent protein kinase (DNA-PK) encapsulates free DNA ends, forming a recruitment platform for downstream end-joining factors including Ligase 4 (LIG4)1. DNA-PK can also bind telomeres and regulate their resection2-4, but does not initiate cNHEJ at this position. How the end joining process is regulated in this context-specific manner is currently unclear. Here we show that the shelterin components TRF2 and RAP1 form a complex with DNA-PK that directly represses its end joining function at telomeres. Biochemical experiments and cryo-electron microscopy reveal that when bound to TRF2, RAP1 establishes a network of interactions with KU and DNA that prevents DNA-PK from recruiting LIG4. In mouse and human cells, RAP1 is redundant with the Apollo nuclease in repressing cNHEJ at chromosome ends, demonstrating that the inhibition of DNA-PK prevents telomere fusions in parallel with overhang-dependent mechanisms. Our experiments show that the end joining function of DNA-PK is directly and specifically repressed at telomeres, establishing a molecular mechanism for how individual linear chromosomes are maintained in mammalian cells.

biochemistry↗

bioRxiv2020

The eukaryotic replisome requires an additional helicase to disarm dormant replication origins

Origins of eukaryotic DNA replication are licensed during G1 phase of the cell cycle by loading the six related minichromosome maintenance (MCM) proteins into a double hexameric ring around double-stranded DNA. In S phase, some double hexamers (MCM DHs) are converted into active CMG (Cdc45-MCM-GINS) helicases which nucleate assembly of bidirectional replication forks. The remaining unfired MCM DHs act as dormant origins to provide backup replisomes in the event of replication fork stalling. The fate of unfired MCM DHs during replication is unknown. Here we show that active replisomes cannot remove unfired MCM DHs. Instead, they are pushed ahead of the replisome where they prevent fork convergence during replication termination and replisome progression through nucleosomes. Pif1 helicase, together with the replisome, can remove unfired MCM DHs specifically from replicating DNA, allowing efficient replication and termination. Our results provide an explanation for how excess replication license is removed during S phase.

biochemistry↗

bioRxiv2020

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

molecular biology↗