ProbingE. coliSSB Protein-DNA topology by reversing DNA backbone polarity

Abstract

AbstractE. colisingle strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homo-tetramers bind ssDNA in two major modes differing in occluded site size and cooperativity. The (SSB)35mode in which ssDNA wraps on average around two subunits is favored at low [NaCl] and high SSB to DNA ratios and displays high “unlimited”, nearest-neighbor cooperativity forming long protein clusters. The (SSB)65mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (> 200 mM) and displays “limited” low cooperativity. Crystal structures ofE. coliSSB andP. falciparumSSB show ssDNA bound to the SSB subunits (OB-folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examinedEcSSB andPfSSB binding to a series of (dT)70constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse polarity (RP) phosphodiester linkage, either 3’-3’ or 5’-5’. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)70with a 3’-3’ polarity switch shows decreased affinity forEcSSB in the (SSB)65mode and lower cooperativity in the (SSB)35mode. However, (dT)70in which every phosphodiester linkage is reversed, does not form a completely wrapped (SSB)65mode, but rather bindsEcSSB in the (SSB)35mode, with little cooperativity. In contrast,PfSSB, which binds ssDNA only in an (SSB)65mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB-folds if there is a change in ssDNA wrapping topology of the RP ssDNA.Statement of SignificanceSingle stranded (ss) DNA binding (SSB) proteins are essential for genome maintenance. Usually homo-tetrameric, bacterial SSBs bind ssDNA in multiple modes, one of which involves wrapping 65 nucleotides of ssDNA around all four subunits. Crystal structures ofE. coliandP. falciparumSSB-ssDNA complexes show ssDNA bound with different backbone polarity orientations raising the question of whether these SSBs maintain strict backbone polarity in binding ssDNA. We show that bothE. coliandP. falciparumSSBs can still form high affinity fully wrapped complexes with non-natural DNA containing internal reversals of the backbone polarity. These results suggest that both proteins maintain a strict backbone polarity preference, but adopt an alternate ssDNA wrapping topology.