Bacterial SSB

E. coli SSB (EcoSSB) is arguably the most intensively studied SSB with many extensive reviews detailing its structure, functions and roles.76 It displays a well characterised homotetramer structure with each 19 kDa monomer consisting of a single OB fold, surrounded by extended loops that support contacts between the monomers and other tetramers in addition to guiding the ssDNA around the tetramer.32, 77, 78 Each monomer also has an acidic C terminal tail that has been shown to be involved in protein recruitment, and also has a possible role in binding to ssDNA although there are conflicting reports to the extent of its contribution.78-81 The homotetramer structure is shown in Figure 1.9 which also displays the four intrinsically disordered C terminal tails and the sequence of the acidic tips.

Similar to RPA, the multiple sites EcoSSB employs to bind to ssDNA allow the diffusion and re-distribution of tetramers in a nucleofilament. Coupled with many possible protein-protein interactions, this allows EcoSSB to assist in many different roles in the cell. EcoSSB can wrap ssDNA around all four of its monomers, using multiple points along the surface of EcoSSB both in and around the OB folds.32 These contacts provide both electrostatic and base stacking interactions, contributing to a strong overall affinity towards the ssDNA. EcoSSB has multiple binding modes that are dependent on monovalent salt concentration, pH, divalent and multivalent

cation concentrations, temperature and protein concentrations, with the (SSB)35 and

(SSB)65 modes being the two most prevalent, which are shown in Figure 1.10.82, 83

The number of nucleotides occluded is dependent on whether two or four monomers are involved in binding, with each respective mode binding to 35 and 65 nt, thus giving the different modes their name. The (SSB)35 mode is favoured under low salt

conditions and high EcoSSB concentrations, showing highly cooperative binding between tetramers along a strand of ssDNA.84 The (SSB)65 mode has a limited

cooperativity producing beads consisting of two tetramers bound on ssDNA, resulting from low protein concentrations and high salt concentrations.32

Figure 1.9: A cartoon showing the tetrameric structure of EcoSSB bound to ssDNA.

The four monomers are coloured blue, green, yellow and white with the four unstructured C terminal tail modelled as grey ribbons and the acidic C terminal residues are shown in red. The ssDNA in the (SSB)65 binding mode is represented as a red ribbon wrapped around the tetramer. Figure adapted

from Kozlov et al.85

The interaction of SSB with other proteins as well as ssDNA is clearly advantageous, yet modifications to an OB fold to achieve this must be done without destabilising the hydrophobic core that is occupied with providing a strong interaction with the

ligand. Loops that act as linkers between β sheets are more likely to be involved in ssDNA contacts and with neighbouring SSB monomers in the SSB filament.

Many SSBs have developed an intrinsically disordered C terminal tail that is characteristically acidic towards its extreme end, including EcoSSB which has one unstructured tail per monomer shown in Figure 1.9.31, 81, 86, 87 This hydrophilic tail is thought to be able to recruit other proteins to ssDNA by protruding out from the SSB as it is bound to ssDNA, which provides a surface for an electrostatic interaction with positively charged areas of other proteins that are involved in DNA processes, such as transcription, replication and DNA repair.26, 29, 86, 88 For example, EcoSSB interacts directly with the clamp loader within DNA polymerase III holoenzyme (Pol III HE), which assists clamp loading, aids processivity and allows the efficient removal of tetramers from ssDNA that could potentially upset the polymerases efficiency during DNA replication.89 EcoSSB’s association with E. coli primase strengthens the primase’s interaction with the nascent RNA primer.90

Dissociation of EcoSSB from the primase also destabilises the primase’s hold on ssDNA, and allows the clamp assembly to occur. During DNA recombination, EcoSSB stimulates RecQ helicase activity via interactions with the EcoSSB C terminal tail.91 EcoSSB also has a role in stabilising the binding and promoting the activities of the exonuclease RecJ,92 and the RecG helicase,93 as well as mediating the formation of RecA filaments through interactions with RecO.94

Figure 1.10: A cartoon showing two of EcoSSBs binding modes.

EcoSSB is shown binding to ssDNA where (a) wraps 65 nt ssDNA around all four monomers and (b) where only 35 nt ssDNA is wrapped around two EcoSSB monomers. (c) The fully cooperative binding of three tetramers (the middle tetramer has its four monomers colour coded to match the tetramers in (a) and (b)) to ssDNA produced by (SSB)35, with strong interactions between proteins

assisted by the L45 loop. Figure modified from Raghunrathan et al.32

In DNA repair processes, EcoSSB recruits the exonuclease E. coli ExoI during MMR,95 and interacts with Uracil DNA glycosylase during BER.96 DNA polymerase II (pol II) is involved in a variety of responses to damaged DNA and requires EcoSSB to process efficiently along DNA and to stimulate pol II-associated nuclease activity.97 Pol II was the first protein to be identified that interacted with EcoSSB,

being co-purified in 1972.26 Their interaction is also shown by the formation of a pol II/SSB complex in the absence of ssDNA.22, 97