Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein

Abstract

Improper maintenance of the mitochondrial genome progressively disrupts cellular respiration and causes severe metabolic disorders commonly termed mitochondrial diseases. Mitochondrial single-stranded DNA binding protein (mtSSB) is an essential component of the mtDNA replication machinery. We utilized single-molecule methods to examine the modes by which human mtSSB binds DNA to help define protein interactions at the mtDNA replication fork. Direct visualization of individual mtSSB molecules by atomic force microscopy (AFM) revealed a random distribution of mtSSB tetramers bound to extended regions of single-stranded DNA (ssDNA), strongly suggesting non-cooperative binding by mtSSB. Selective binding to ssDNA was confirmed by AFM imaging of individual mtSSB tetramers bound to gapped plasmid DNA substrates bearing defined single-stranded regions. Shortening of the contour length of gapped DNA upon binding mtSSB was attributed to DNA wrapping around mtSSB. Tracing the DNA path in mtSSB–ssDNA complexes with Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy established a predominant binding mode with one DNA strand winding once around each mtSSB tetramer at physiological salt conditions. Single-molecule imaging suggests mtSSB may not saturate or fully protect single-stranded replication intermediates during mtDNA synthesis, leaving the mitochondrial genome vulnerable to chemical mutagenesis, deletions driven by primer relocation or other actions consistent with clinically observed deletion biases.

Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase

“…Previously, we utilized AFM to examine the binding modes of E. coli SSB and human mtSSB to ssDNA. Unlike E. coli SSB, mtSSB does not form nucleoprotein filaments that are a hallmark of cooperative DNA binding but instead binds non-cooperatively to extended ssDNA structures at physiological concentrations of salt (53). The current study utilizes similar solution conditions, and nucleoprotein filaments were not observed in any of our AFM images.…”

“…Analysis of the fractional occupancies (39) of Twinkle along the linear gapped DNA establishes that Twinkle has a high binding specificity (S=KSP/KNSP, ~2.5 x 10 3 ) for the ssDNA gap. In comparison, mtSSB displays a higher binding specificity (S=KSP/KNSP = 1.45 x 10 4 ) determined by the same method (53). Highly specific binding of Twinkle to the ssDNA gap suggested this defined DNA substrate is ideally suited to assess DNA unwinding at the singlemolecule level.…”

“…Diagnostic restriction digestion of the gapped region indicated typical DNA gapping efficiencies of 85 to 95% (53,71). Linear dsDNA lacking a ssDNA gap was prepared by directly linearizing pSCW01 plasmid DNA with ScaI restriction endonuclease.…”

Mammalian mitochondrial DNA replication and mechanisms of deletion formation

“…The precise mode of mtSSB binding is still under investigation. Conflicting reports have provided evidence for both cooperative and non-cooperative binding (Wong TS et al 2009;Miralles Fuste et al 2014;Qian and Johnson 2017;Kaur et al 2018).…”

Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis

“…Sequence comparison showed that the major differences between these two proteins are that residues 1–9 and 55–59 of mtSSB are missing in EcoSSB and that EcoSSB contains an additional C-terminal tail missing in mtSSB (11). Apart from these differences, these two SSBs share fast binding (10 9 –10 11 M −1 s −1 ) and slow dissociation (10 −1 –10 −2 s −1 ) kinetics to and from ssDNA (12,13) and bind preformed ssDNA as tetramers in at least two different modes. In each mode, ∼30 (mtSSB 30 ) or ∼60 (mtSSB 60 ) nucleotides are organized per tetramer depending on the salt and protein concentrations (3,12–14).…”