Mersch, K. | bioRxiv Science

bioRxiv2022

Helicase Activity Promoted Through Dynamic Interactions Between a ssDNA Translocase and a Diffusing SSB Protein

Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches we show that S. cerevisiae Pif1 can use its ATP-dependent 5 to 3 translocase activity to chemo-mechanically push a single human RPA (hRPA) directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a new mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein.

biophysics↗

bioRxiv2020

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

CLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of -10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

biophysics↗