To investigate potential movements of SSB on ssDNA, we employed single molecule fluorescence resonance energy transfer (smFRET)8,9
. FRET efficiencies E
from individual immobilized partial duplex DNA with a 3’ (dT)N
tail (64 ≤ N ≤ 131) bound to SSB were acquired using total internal reflection fluorescence microscopy9
. Surface immobilization and fluorescent labelling have no measurable effect on the dynamics of SSB binding mode transitions5
. Owing to the closed wrapping in the (SSB)65
binding mode favoured under our conditions (500 mM NaCl or 10 mM Mg2+
, when SSB is bound to ssDNA of 65-70 nt with its two ends labelled with donor (Cy3) and acceptor (Cy5) fluorophores, singular high FRET distributions were observed5
. However, when a (dT)69
tail is further extended by an additional 12 nt of sequence complementary to the overhanging cohesive end of l-strand of λ phage DNA, individual SSB-ssDNA complexes display large FRET fluctuations in the millisecond time scale (). These fluctuations were dramatically suppressed when the 12 nt extension is hybridized to a cohesive end of a λ DNA (). To exclude binding and dissociation of additional SSB molecules as the cause of fluctuations, unbound SSB was removed by a buffer wash before measurements. DNA unwrapping/rewrapping dynamics, occurring in tens of microseconds in high salt3,4
, is completely averaged out within our 10-30 ms time resolution5
. We also ruled out local melting of the duplex portion as a source of fluctuations (Supplementary materials, SM1
). Therefore, these fluctuations must arise from additional conformational states enabled by the 12 nt extension.
FRET fluctuations arising from diffusional migration of SSB on ssDNA
To test whether the FRET fluctuations are caused by transient excursions of SSB to the extension, we varied the length of the extension ((dT)N
, N= 0 - 18) while keeping the ssDNA between Cy3 and Cy5 at 69 or 70 nt. If an SSB tetramer binds randomly and remains fixed at the initial site of binding undergoing only transient interactions with ssDNA outside the binding site, each complex will generate a FRET distribution that is unique to the initial site of binding. However, all complexes for each construct displayed similar FRET time trajectories (Supplementary Fig. 1
). Furthermore, if SSB migrates along the DNA, larger excursions away from the high FRET state are expected for longer extensions. Indeed, average FRET values decreased for longer extensions while the high FRET state was still transiently visited (Supplementary Fig. 1
). The FRET distribution and the time scale of fluctuations are relatively independent of the salt concentration (Supplementary Fig. 2
), arguing against these FRET changes arising from binding mode transitions which display a strong salt dependence10-12
. Hence, these fluctuations likely reflect SSB's diffusional migration on ssDNA with the different FRET values corresponding to different SSB locations.
To make unbiased assignments of FRET states, we employed a hidden Markov model (HMM) based statistical approach that determines the most likely time sequence of FRET states () 13,14
. The result is further reduced to a transition density plot (TDP)13,14
, that allows the number of distinct FRET states, their FRET values, and the transition rates to be estimated (). We analyzed SSB migration on DNA molecules with several 3’ dT tail lengths (0 to 12 nt extension beyond 65 nt binding site size) at 13 °C to slow down migration (). Longer extensions gave multiple indistinguishable low FRET states in the TDP (Supplementary Fig. 3
). For (dT)69+8
(12 nt extension from the 65 nt binding site size with 69 nt separation between fluorophores), six distinct FRET states were resolved () with transitions occurring between nearest neighbours. We assigned the highest FRET value (E
~ 0.8) to the state with SSB closest to the ss-dsDNA junction and lower FRET values for positions away from the junction. The rates of transition, or the ‘stepping rates’, were independent of the beginning and ending state of transition (Supplementary Fig. 4
) and ranged between 3.0 and 4.5 s−1
(). Similar analysis yielded 5, 3 and 2 states for DNA with 8, 2 and 0 nt extensions, respectively (Supplementary Fig. 3
). Therefore, every 2-4 nt of DNA extension provides an additional configuration, yielding an apparent step size of about 3 nt.
Analysis of SSB mobility on ssDNA
Because FRET fluctuations became too fast for HMM analysis above 13°C, we used autocorrelation analysis of FRET efficiency E for the temperature dependence studies (). The averaged auto-correlation function plots of the SSB-(dT)69+8 complexes were best fit by bi-exponential decays. The shorter lifetime was equal to the time resolution independent of temperature and is ascribed to photophysical or detection noise The longer lifetime, τlong, displayed a monotonic temperature dependence and was attributed to SSB diffusion. The Arrhenius fit of ln(1/τlong) vs. 1/T () gave an apparent activation energy of 81 ± 7 kJ/mol. Combined with the stepping rate of ~ 4 s−1 at 13 °C, we can then estimate a stepping rate of ~ 60 s−1 at 37 °C. Assuming a 3 nt step size, the diffusion coefficient of an SSB tetramer along ssDNA at 37 °C is estimated to be 270 (nt)2/s.
As a further test of SSB migration on the ssDNA, we employed single molecule 3 color FRET9,15
using a donor-labelled SSB mutant (A122C labelled with ~1 Alexa555 per SSB tetramer) and two different acceptors, Cy5 and Cy5.5, attached to the two ends of a (dT)130
(). The large separation between the two acceptors eliminates any significant FRET between them. If a single SSB tetramer diffuses on the long ssDNA, high FRET events to either acceptor will be mutually exclusive. Indeed, we observed rapid and anti-correlated fluctuations of apparent FRET efficiencies to the two acceptors, Eapp
, demonstrating that SSB truly diffuses on the DNA (). To ensure single SSB molecules on DNA, 1 min incubation with sub-saturating concentrations of SSB (< 100 pM) was followed immediately with a buffer wash and only traces displaying single donor photobleaching events were analyzed. At higher SSB concentration (10 nM), much slower FRET fluctuations were observed likely due to binding of additional SSB (Supplementary Figure 5
SSB diffusion on ssDNA probed with three-color FRET
To probe how far SSB can move on a long ssDNA, we placed Cy5 and Cy5.5 on the two ends of a (dT)130 and Cy3 in the middle (named (dT)65+65). This 3-color FRET scheme allows us to determine at which end the SSB was present by following the ‘closed’ wrapping of that DNA segment and high FRET to the corresponding acceptor (). Both the dye pairs display transient high FRET states that are anti-correlated indicating that the same SSB molecule was capable of migrating to either end of the DNA (). Therefore, SSB can move at least 65 nt via diffusion and is not constrained to its initial binding site.