DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein

Abstract

Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB–DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during ‘in situ’ DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.

Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers

“…The great flexibility of ssDNA and large heterogeneity of the protein-DNA interface has hindered the advance of the research in this field. The model and the associated method described here can be applied beyond the disperse ligand regime, as shown in single molecule manipulation studies with the HmtSSB [61]. …”

“…Fitting the experimental force extension curves to these equations allows determining the coverage and the binding mode when the effective size of the ligand is known, as we have recently shown for human mitochondrial SSB protein [61]. …”

“…(Panel B): Equilibrium extension of the covered polymer as function of the pulling force, resulting from employing the coverage of Panel A in the force-extension relation Eq (9). In this figure the values of the parameters of the polymer are of the order of those of ssDNA [24]: N = 5080, d 0 = 0.57 nm , L p = 0.715 nm , K 0 = 700 pN and K B T = 4.11 pN nm , while, for the ligands, we consider two different binding modes, with parameters of the order of those of E. Coli SSB [47,59] and HmtSSB proteins [61]: m = 65 or 35, a = 5 nm and ϵ b = m × 0.5 × K B T .…”

“…Also in Fig 4B we combine this result with the mechanical model in Section II and represent the corresponding force extension curves for a particular case with similar parameters to the single strand binding protein (SSB) bound to single strand DNA (ssDNA), for the particular cases of E. Coli SSB [47,59] and human mitochondrial SSB [61]. These plots have the same form as those experimentally obtained for these two SSB in recent single-molecule experiments [47,61]. It is important also to notice that Tonks model predicts a maximum coverage lower than the complete coverage, see Fig 4A.…”

Self-regulation of single-stranded DNA wrapping dynamics byE. coliSSB promotes both stable binding and rapid dissociation

“…Here we consider the case of genomic replication with the assumption that the template ssDNA is saturated with EcSSB in an intermediately wrapped state such as EcSSB35, as was previously seen with the human mitochondrial SSB that is structurally and functionally similar to EcSSB (43). However, we must also note that EcSSB exists in a dynamic equillibrium between its distinct modes that are able to diffuse along the DNA without dissociation (29,43). Therefore, a processing enzyme, the DNA polymerase (DNA pol) in this case, may displace the wrapped EcSSB during synthesis, pushing it forward along the template strand.…”