Structural Insights into Bacterial Helicase Loading at a Replication Origin by Cryogenic Electron Microscopy

Author

Abhipsa Shatarupa, The Graduate Center, City University of New YorkFollow

Date of Degree

9-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

David Jeruzalmi

Committee Members

Amedee des Georges

Anuradha Janakiraman

Brian Chait

Jonathan Dworkin

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Keywords

Structural Biology, Cryo-EM

Abstract

DNA replication is a meticulously regulated and intricate process crucial for cellular viability. Replicative helicases, essential for unwinding DNA during replication, rely on helicase loader proteins for efficient loading and assembly at replication origins. In Escherichia coli, the replicative helicase DnaB forms a complex with six copies of the bacteriophage Lambda λP loader, resulting in a B₆P₆ conformation. The conformation of the BP complex, with a 6:6 stoichiometry, indicates that the isolated DnaB is initially captured by λP (B₆P₆) through its carboxy-terminal domain lassos/grappling hooks, serving as an early intermediate before transitioning into an open spiral conformation. This complex gradually transitions into a right-handed open spiral configuration, facilitated by the displacement of one out of the six λP protomers, resulting in a B₆P₅ stoichiometry of the complex. The formation of this open spiral creates an internal chamber accessible to physiological single-stranded DNA (ssDNA). In the high-resolution 2.66 Å and 2.84 Å models of the DnaB-λP complex, with a 6:5 stoichiometry (B₆P₅), the pentameric λP ensemble adopts an asymmetric configuration, with one λP copy binding at the top and bottom of the open DnaB spiral. Although the internal chamber of DnaB remains accessible to ssDNA, the λP protomer spans the breach and covers the top tier of the BP complex. Additionally, domains II of two more copies of λP effectively blocks the path from the top tier of BP into the inner chamber, resulting in an autoinhibited configuration for the complex. Nonetheless, despite this obstruction, the BP complex is capable of binding ssDNA, and we suggest that the path for ssDNA lies outside the DnaB ring. These conformational changes in the helicase-loader complex suggests that the complex can now be recruited to the initiator protein-DNA complex at the Phage replication origin and continue the process of replication initiation. Additionally, comparisons between the λP and E. coli DnaC complexes of the DnaB helicase offer new insights into how these two loaders, despite their structural differences, converge on the same mechanism of ring opening. These findings deepen our understanding of the intricate processes underlying DNA replication initiation mechanisms.

Recommended Citation

Shatarupa, Abhipsa, "Structural Insights into Bacterial Helicase Loading at a Replication Origin by Cryogenic Electron Microscopy" (2024). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/5972