Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

48-POS Board 48 SELF-REGULATION OF SINGLE-STRANDED DNA WRAPPING DYNAMICS BY E. COLI SSB MEDIATED THROUGH COMPETITIVE BINDING. Nabuan Naufer 1 ; Michael Morse 1 ; Ioulia F Rouzina 2 ; Penny J Beuning 3 ; Mark C Williams 1 ; 1 Northeastern University, Physics, Boston, MA, USA 2 Ohio State University, Biochemistry, Columbus, OH, USA 3 Northeastern University, Chemistry and Chemical Biology, Boston, MA, USA Single-stranded DNA (ssDNA) binding proteins (SSBs) rapidly bind and protect transiently formed ssDNA intermediates during genomic maintenance and replication processes. E. coli SSB (EcSSB), a homotetramer that wraps the ssDNA in multiple topologies, is a model protein used to study SSB function. Here, we characterize the collective binding dynamics of EcSSB to a long ssDNA template using optical tweezers. We show that EcSSB binds to ssDNA in a biphasic manner where EcSSB initially wraps the ssDNA, and then unwraps from the ssDNA as the substrate becomes saturated with protein. A general two-step kinetic model in which the binding state of EcSSB is regulated through ssDNA occupancy describes the observed competitive binding dynamics. Our results uncover an unwrapped SSB binding mode that is confirmed using AFM imaging. In this mode EcSSB binds ssDNA segments as short as 8 nt and is thus signified as (SSB) 8 . Furthermore, while EcSSB slowly dissociates from ssDNA through increased ssDNA tension, salt concentration, and displacement by RecA filaments, EcSSB wrapping stimulates rapid dissociation of neighboring proteins on a saturated ssDNA substrate. Taken together, our results suggest a mechanism by which tightly wrapped EcSSB dissociate from ssDNA to self- regulate the protein density ensuring its transient role during genomic maintenance and replication. In addition, the proposed mechanism provides insights on biological implications of the distinct EcSSB binding topologies significantly enhancing the understanding of SSB function.

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