PROJECT SUMMARY/ABSTRACT When a replication fork encounters a DNA lesion in a template strand, replication gives way to DNA repair and recombination. These encounters define an interface in DNA metabolism that can give rise to genome instability. This is ultimately manifested in tumor evolution in eukaryotes and the development of antibiotic resistance and increased pathogenicity in bacteria. In this renewal application, we are investigating perhaps the most enigmatic of repair processes, the repair of lesion-containing post-replication gaps. When a replisome encounters a template lesion, disengages, and then re-initiates upstream, the lesion is left behind in a post- replication gap. The existence of these gaps has been appreciated for over 5 decades, but progress has been limited by methodology that has been inadequate to properly explore their general importance and repair. These gaps are primary substrates for DNA synthesis by translesion DNA polymerases, recombinational DNA repair, and replicational template switching, all processes linked with genomic instability. Work in the last funding period has featured the development of a range of essential new methods, as well as conceptual advances in our understanding of how post-replication gaps are generated and processed. In E. coli, we now know that post-replication gaps are generated several times each replication cycle and that gap formation is triggered by encounters with bulky nucleotide lesions. We have laid out the pathways by which the gaps are targeted and resolved by particular DNA repair proteins. We are now in a position to provide a deep molecular understanding of these pathways. As a bonus, we have identified four potential bacterial vulnerabilities that may eventually provide pharmaceutical advances to treat antibiotic-resistand pathogens or slow the development of antibiotic resistance. We bring together world-class expertise in biochemistry, genetics, molecular biology, and biophysics. We will apply our new methods, including novel single-molecule approaches, towards detecting and quantifying gaps and further characterize the proteins acting on them. While driven by our mechanistic questions, the new methods will broadly benefit research in genomic maintenance. The six specific aims constitute a systematic attack on the problem. Aims 1-3 focus on the recombinational DNA repair of post-replication gaps by the RecFOR system. Aim 4 is an exploration of the single-stranded DNA binding protein (SSB) and how it directs the division of labor in gaps via its interactions with 20 or more different repair proteins. Aim 5 focuses on the gap-related activation of the mutagenic DNA polymerase V and its homologues, the source of most mutagenesis in any bacterial cell. Finally, aim 6 investigate an entirely new biological phenomenon, the presence of DNA sequence elements in the bacterial genome that force the formation of post-replication gaps in particular locations.