Single Stranded DNA Translocases can Push Single Stranded DNA Binding Proteins along Single Stranded DNA

Abstract

Single-stranded (ss) DNA binding proteins (SSBs) protect ssDNA intermediates during genome maintenance. These SSBs must eventually be displaced or reorganized and one potential mechanism for reorganization is pushing of the SSB along the DNA by ssDNA translocases (ATP-dependent motors). Single molecule FRET total internal reflection fluorescence (smTIRF) microscopy allows for observation of such phenomena that would otherwise be averaged out in ensemble experiments. When Cy5-labeled E. coli SSB or human replication protein A (RPA) is bound to 3’-Cy3 labeled ssDNA immobilized to the slide surface, anti-correlated donor and acceptor fluctuations are seen, consistent with a bound SSB diffusing randomly along the ssDNA. For both EcSSB and hRPA, introduction of S. cerevisiae Pif1, a SF1 5’ to 3’ ssDNA translocase/helicase, in the presence of ATP causes a decrease in the number of diffusing SSB molecules observed and an appearance of isolated, irregularly spaced ‘saw-tooth’ FRET signals. These asymmetric FRET spikes reflect the translocase pushing the acceptor-labeled SSB towards the donor-labeled 3’ end of the ssDNA followed by dissociation of the SSB. This translocase-induced effect is ATP dependent and is not observed with the addition of Pif1 alone, indicating that SSB clearance and ‘pushing’ are due to Pif1 translocation and not simply Pif1 binding. Simulations of translocase-induced directed motion of the SSB indicate that the ATP dependence observed is more consistent with an active pushing mechanism rather than passive rectification of SSB diffusion. Similar pushing events are observed on 5′-Cy3 labeled ssDNA with E. coli UvrD and Rep (both 3′ to 5′ ssDNA translocases). These results provide direct evidence for translocase-induced pushing of SSB proteins along ssDNA as a potential mechanism for SSB reorganization and clearance. (TML: NIH GM030498/GM045948, RG: NIH GM098509, JES: ACS 127658).