The Escherichia coli single-strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single-stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide-oligonucleotide binding folds (OB-fold) present in SSB and its interactome partners. Not surprisingly, partner OB-fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB-fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C-terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles.First, and consistent with the proposal of Dixon, to regulate the structure of the C-terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB-folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB-folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB-fold genome guardians identified in prokaryotes. K E Y W O R D SDNA helicase, OB-fold, PXXP motif, RecG, SH3 domain, single-strand binding protein, SSB interactome

The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB-interactome. Key to both roles is the C-terminal, one-third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target. In this study, they examine the role of the linker region in SSB function in a variety of DNA metabolic processes in vitro. Using the same linker mutants, the results show that in addition to association reactions (either DNA or protein), the IDL is critical for the release of SSB from DNA. This release can be under conditions of ssDNA competition or active displacement by a DNA helicase or recombinase. Consistent with their previous work these results indicate that SSB linker mutants are defective for SSB-SSB interactions, and when the IDL is removed a terminal SSB-DNA complex results. Formation of this complex inhibits downstream processing of DNA by helicases such as RecG or PriA as well as recombination, mediated by RecA. A model, based on the evidence herein, is presented to explain how the IDL acts in SSB function.

To remain globally competitive, education in the United States must focus on retaining students in science, technology, engineering, and mathematics (STEM). As laboratory courses have the potential to be powerful attractors or deterrents to a field, developing effective laboratory pedagogies is important to retain students in STEM. A course-based undergraduate research experience (CURE) is one model for laboratory instruction that has generated an increasing amount of attention. This has been driven by their success, especially in increasing students’ self-efficacy and science identity, two long-term indicators of persistence in STEM. Herein is reported a large introductory organic chemistry CURE course framework that focuses on green reaction optimization. Students were given an unoptimized alkene epoxidation procedure, split into small “research” groups, and tasked with improving conversion while minimizing reaction inputs. After three rounds of optimization, groups increased conversion 10–20-fold compared to baseline conditions. Then, students investigated the substrate scope of their conditions and analyzed trends in relative reactivities based on principles learned in lecture and from the literature. Lastly, students summarized their findings in a final slideshow presentation. Surveys were used to evaluate different aspects common to CURE courses and students’ sense of project ownership. The scores obtained were consistent with other reported CURE courses. Importantly, students saw large gains in self-efficacy and science identity. Students had an overwhelmingly positive response to the curriculum based on informal and written feedback. This approach is generalizable to a wide range of institutions with different equipment availability, reaction types, and course coverage schedules.

Large introductory STEM courses have a reputation as “weed out” classes. These classes and their laboratory components are more often viewed by students as hurdles to overcome than valuable learning experiences relevant to their future careers. This perception especially impacts students who have a lower science identity and self-efficacy compared to their peers, which is often true for students who identify as being from groups historically underrepresented in STEM or first-generation college students. In Fall 2020, the curriculum for a second-semester organic chemistry course was redesigned as an entirely remote, inquiry-based course focused on transferable skills. The chemistry explored was epoxidation and epoxide-opening reactions, which students learned about in lectures early in the semester. Students evaluated reaction progress using real and complex data, and they integrated their learning in the form of a final group presentation. The emphasis of this course was on transferable skills, not specific reactions or laboratory techniques. The latter were used as vehicles by which to teach students how to analyze data, interpret results, and develop their communication skills. Student survey responses demonstrated that this remote course helped all students increase their self-efficacy and science identity. However, in just one semester, the pre-existing gaps in self-efficacy and science identity between students from underrepresented groups (historically underrepresented groups and first-generation college students) and their peers closed entirely. Taken together, these results suggest the incredible value of an inquiry-based laboratory curriculum, even in a remote course. This is a particularly valuable approach in large introductory courses, which are a site of significant attrition in STEM career pathways.