Biology subjects

Bell, S. P.

Publications and source records attributed to Bell, S. P..

3 recordsLinked to original sources

bioRxiv2022

Distinct RPA functions promote eukaryotic DNAreplication initiation and elongation

Single-stranded DNA binding proteins (SSBs) are essential for DNA replication across all domains of life, but vary significantly in their structure and subunit composition. The eukaryotic SSB, Replication Protein A (RPA), serves critical functions in DNA replication, the DNA damage response, and DNA repair. We sought to determine the requirements for RPA during eukaryotic DNA replication initiation and elongation. To determine whether the ssDNA-binding activity is sufficient, we tested SSBs from different domains of life in reconstituted S. cerevisiae origin unwinding and DNA replication reactions. Interestingly, E. coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we found that only large, multimeric complexes with multiple DNA-binding domains support origin unwinding. In contrast, our studies demonstrated that eukaryotic replication fork function requires specific RPA domains for normal leading- and lagging-strand DNA synthesis. Together, these results reveal new requirements for ssDNA-binding proteins in eukaryotic replication origin unwinding and uncover RPA domains that are critical for faithful replication fork function.

biochemistry↗

bioRxiv2021

A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2-7 during helicase loading. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

molecular biology↗

bioRxiv2020

DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

The committed step of eukaryotic DNA replication occurs when the replicative Mcm2-7 helicase pairs that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45 and GINS are individually recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). The multiple phosphorylation sites on the Mcm2-7 tails promote DDK-dependent modulation of the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity results in increased frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for the control of replication origin efficiency and timing.

cell biology↗