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1. In vivo fluorescent TUNEL detection of single stranded DNA gaps and breaks induced by dnaB helicase mutants in Escherichia coli

   https://doi.org/10.1016/bs.mie.2022.02.021  

   Behrmann, Megan S; Trakselis, Michael A. (August 2022, Methods in enzymology)

   Trakselis, Michael A. (Ed.)

   The genome of prokaryotes can be damaged by a variety of endogenous and exogenous factors, including reactive oxygen species, UV exposure, and antibiotics. To better understand these repair processes and the impact they may have on DNA replication, normal genome maintenance processes can be perturbed by removing or editing associated genes and monitoring DNA repair outcomes. In particular, the replisome activities of DNA unwinding by the helicase and DNA synthesis by the polymerase must be tightly coupled to prevent any appreciable single strand DNA (ssDNA) from accumulating and amplifying genomic stress. If decoupled, vulnerable ssDNA would persist, likely leading to double strand breaks (DSBs) or requiring replication restart mechanisms downstream of a stall. In either case, free 3'-OH strands would exist, resulting from ssDNA gaps in the leading strand or complete DSBs. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) can enzymatically label ssDNA ends with bromo-deoxy uridine triphosphate (BrdU) to detect free 3'-OH DNA ends in the E. coli genome. Labeled DNA ends can be detected and quantified using fluorescence microscopy or flow cytometry. This methodology is useful in applications where in situ investigation of DNA damage and repair are of interest, including effects from enzyme mutations or deletions and exposure to various environmental conditions. 

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2. C-terminal Domain of T4 gene 32 Protein Enables Rapid Filament Reorganization and Dissociation

   https://doi.org/10.1016/j.jmb.2024.168544  

   Cashen, Ben A; Morse, Michael; Rouzina, Ioulia; Karpel, Richard L; Williams, Mark C (May 2024, Journal of Molecular Biology)

   Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein essential for DNA replication. gp32 forms stable protein filaments on ssDNA through cooperative interactions between its core and N-terminal domain. gp32′s C-terminal domain (CTD) is believed to primarily help coordinate DNA replication via direct interactions with constituents of the replisome. However, the exact mechanisms of these interactions are not known, and it is unclear how tightly-bound gp32 filaments are readily displaced from ssDNA as required for genomic processing. Here, we utilized truncated gp32 variants to demonstrate a key role of the CTD in regulating gp32 dissociation. Using optical tweezers, we probed the binding and dissociation dynamics of CTD-truncated gp32, *I, to an 8.1 knt ssDNA molecule and compared these measurements with those for full-length gp32. The *I-ssDNA helical filament becomes progressively unwound with increased protein concentration but remains significantly more stable than that of full-length, wild-type gp32. Protein oversaturation, concomitant with filament unwinding, facilitates rapid dissociation of full-length gp32 from across the entire ssDNA segment. In contrast, *I primarily unbinds slowly from only the ends of the cooperative clusters, regardless of the protein density and degree of DNA unwinding. Our results suggest that the CTD may constrain the relative twist angle of proteins within the ssDNA filament such that upon critical unwinding the cooperative interprotein interactions largely vanish, facilitating prompt removal of gp32. We propose a model of CTD-mediated gp32 displacement via internal restructuring of its filament, providing a mechanism for rapid ssDNA clearing during genomic processing. 

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3. Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

   https://doi.org/10.3390/ijms231911404  

   Sun, Zhiqiang; Stormberg, Tommy; Filliaux, Shaun; Lyubchenko, Yuri L. (October 2022, International Journal of Molecular Sciences)

   Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes. 

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4. Akaby—Cell-free protein expression system for linear templates

   https://doi.org/10.1371/journal.pone.0266272  

   Sato, Wakana; Sharon, Judee; Deich, Christopher; Gaut, Nathaniel; Cash, Brock; Engelhart, Aaron E.; Adamala, Katarzyna P. (April 2022, PLOS ONE)

   Perez-Fernandez, Jorge (Ed.)

   Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based onEscherichia coli(E.coli). Endogenous nucleases inE.colicell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity inE.coli, with the RecB subunit possessing the actual nuclease activity. We created aRecBknockout of anE.colistrain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL sourceE.colistrain, enabling the creation of TXTL systems with other custom modifications. 

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5. Precision Modification of Native Antibodies

   https://doi.org/10.1021/acs.bioconjchem.1c00342  

   Wu, Kuan-Lin; Yu, Chenfei; Lee, Catherine; Zuo, Chao; Ball, Zachary T.; Xiao, Han (August 2021, Bioconjugate Chemistry)

   null (Ed.)

   Antibodies, particularly of the immunoglobulin G (IgG) isotype, are a group of biomolecules that are extensively used as affinity reagents for many applications in research, disease diagnostics, and therapy. Most of these applications require antibodies to be modified with specific functional moieties, including fluorophores, drugs, and proteins. Thus, a variety of methodologies have been developed for the covalent labeling of antibodies. The most common methods stably attach functional molecules to lysine or cysteine residues, which unavoidably results in heterogeneous products that cannot be further purified. In an effort to prepare homogeneous antibody conjugates, bioorthogonal handles have been site-specifically introduced via enzymatic treatment, genetic code expansion, or genetically encoded tagging, followed by functionalization using bioorthogonal conjugation reactions. The resulting homogeneous products have proven superior to their heterogeneous counterparts for both in vitro and in vivo usage. Nevertheless, additional chemical treatment or protein engineering of antibodies is required for incorporation of the bioorthogonal handles, processes that often affect antibody folding, stability, and/or production yield and cost. Accordingly, concurrent with advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in site-specifically labeling native (nonengineered) antibodies without chemical or enzymatic treatments. In this review, we highlight recent strategies for producing site-specific native antibody conjugates and provide a comprehensive summary of the merits and disadvantages of these strategies. 

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