When duplex DNA is altered in almost any way (replicated, recombined, or repaired), single strands of DNA are usually intermediates, and single-stranded DNA binding (SSB) proteins are present. These proteins have often been described as inert, protective DNA coatings. Continuing research is demonstrating a far more complex role of SSB that includes the organization and/or mobilization of all aspects of DNA metabolism. Escherichia coli SSB is now known to interact with at least 14 other proteins that include key components of the elaborate systems involved in every aspect of DNA metabolism. Most, if not all, of these interactions are mediated by the amphipathic C-terminus of SSB. In this review, we summarize the extent of the eubacterial SSB interaction network, describe the energetics of interactions with SSB, and highlight the roles of SSB in the process of recombination. Similar themes to those highlighted in this review are evident in all biological systems.The genomes of all cellular organisms are organized as double-stranded (ds) DNA, with the information content, in the form of nucleotide bases, sequestered in the interior of the protective double helix 1; 2 . To provide DNA replication, recombination, and repair machinery access to genomic information, dsDNA must be unwound to form single-stranded (ss) intermediates. Such processes are obligatory, but they are not without risks. ssDNA is prone to chemical and nucleolytic attacks that can produce breaks or lesions that are difficult to repair and can selfassociate to create impediments to genome maintenance 3-9 . To help preserve ssDNA intermediates, cells have evolved a specialized class of ssDNA-binding (SSB) proteins that associate with ssDNA with high affinity and in a sequence-independent manner 10-21 . SSB binding protects ssDNA from degradation 10; 22-25 , and, more globally, defines the nucleoprotein substrates upon which DNA replication, recombination, repair, and replication restart processes must act. With these central roles in genome maintenance, it is no surprise that SSB proteins are conserved throughout all kingdoms of life and are indispensable for cell survival 26-28 .Beyond their eponymous roles in DNA binding, SSB proteins have a second, less wellappreciated role in which they physically associate with a broad array of cellular genome maintenance proteins. SSB interaction with heterologous proteins targets enzymes to active genome maintenance sites and, in many cases, stimulates the biochemical activities of SSB's partner proteins. In this review, we focus on several aspects of eubacterial SSB interactions with heterologous proteins. First, we summarize the extent of SSB's interaction network by *To whom correspondence should be addressed.. NIH Public Access Author ManuscriptCrit Rev Biochem Mol Biol. Author manuscript; available in PMC 2009 September 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptdescribing what is known about its many partner proteins. In this section, we focus primarily on E. col...

In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 Å resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

We have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) DNA-binding proteins from Bacillus subtilis , SsbA and SsbB. During transformation, SsbB localizes at the DNA entry pole where it binds and protects internalized ssDNA. The 2.8-Å resolution structure of SsbB bound to ssDNA reveals a similar overall protein architecture and ssDNA-binding surface to that of Escherichia coli SSB. SsbA, which binds ssDNA with higher affinity than SsbB, co-assembles onto SsbB-coated ssDNA and the two proteins inhibit ssDNA binding by the recombinase RecA. During chromosomal transformation, the RecA mediators RecO and DprA provide RecA access to ssDNA. Interestingly, RecO interaction with ssDNA-bound SsbA helps to dislodge both SsbA and SsbB from the DNA more efficiently than if the DNA is coated only with SsbA. Once RecA is nucleated onto the ssDNA, RecA filament elongation displaces SsbA and SsbB and enables RecA-mediated DNA strand exchange. During plasmid transformation, RecO localizes to the entry pole and catalyzes annealing of SsbA- or SsbA/SsbB-coated complementary ssDNAs to form duplex DNA with ssDNA tails. Our results provide a mechanistic framework for rationalizing the coordinated events modulated by SsbA, SsbB and RecO that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.

Bacterial single-stranded DNA (ssDNA)-binding proteins (SSBs) play essential protective roles in genome biology by shielding ssDNA from damage and preventing spurious DNA annealing. Far from being inert, ssDNA/SSB complexes are dynamic DNA processing centers where many different enzymes gain access to genomic substrates by exploiting direct interactions with SSB. In all cases examined to date, the C terminus of SSB (SSB-Ct) forms the docking site for heterologous proteins. We describe the 2.7-Å-resolution crystal structure of a complex formed between a peptide comprising the SSB-Ct element and exonuclease I (ExoI) from Escherichia coli. Two SSB-Ct peptides bind to adjacent sites on ExoI. Mutagenesis studies indicate that one of these sites is important for association with the SSB-Ct peptide in solution and for SSB stimulation of ExoI activity, whereas the second has no discernable function. These studies identify a correlation between the stability of the ExoI/SSB-Ct complex and SSB-stimulation of ExoI activity. Furthermore, mutations within SSB's C terminus produce variants that fail to stimulate ExoI activity, whereas the SSB-Ct peptide alone has no effect. Together, our findings indicate that SSB stimulates ExoI by recruiting the enzyme to its substrate and provide a structural paradigm for understanding SSB's organizational role in genome maintenance.genome maintenance ͉ SSB

Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.X-ray crystal structure | single-molecule FRET