Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli.

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon-insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in double-strand DNA break homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.

Mimicking Natural Photosynthesis: Designing Ultrafast Photosensitized Electron Transfer into Multiheme Cytochrome Protein Nanowires.

Efficient nanomaterials for artificial photosynthesis require fast and robust unidirectional electron transfer (ET) from photosensitizers through charge-separation and accumulation units to redox-active catalytic sites. We explored the ultrafast time-scale limits of photo-induced charge transfer between a Ru(II)tris(bipyridine) derivative photosensitizer and PpcA, a 3-heme c-type cytochrome serving as a nanoscale biological wire. Four covalent attachment sites (K28C, K29C, K52C, and G53C) were engineered in PpcA enabling site-specific covalent labeling with expected donor-acceptor (DA) distances of 4-8 Å. X-ray scattering results demonstrated that mutations and chemical labeling did not disrupt the structure of the proteins. Time-resolved spectroscopy revealed three orders of magnitude difference in charge transfer rates for the systems with otherwise similar DA distances and the same number of covalent bonds separating donors and acceptors. All-atom molecular dynamics simulations provided additional insight into the structure-function requirements for ultrafast charge transfer and the requirement of van der Waals contact between aromatic atoms of photosensitizers and hemes in order to observe sub-nanosecond ET. This work demonstrates opportunities to utilize multi-heme c-cytochromes as frameworks for designing ultrafast light-driven ET into charge-accumulating biohybrid model systems, and ultimately for mimicking the photosynthetic paradigm of efficiently coupling ultrafast, light-driven electron transfer chemistry to multi-step catalysis within small, experimentally versatile photosynthetic biohybrid assemblies.

Characterization of the structure and catalytic activity of Legionella pneumophila VipF.

The pathogenic bacteria Legionella pneumophila is known to cause Legionnaires' Disease, a severe pneumonia that can be fatal to immunocompromised individuals and the elderly. Shohdy et al. identified the L. pneumophila vacuole sorting inhibitory protein VipF as a putative N-acetyltransferase based on sequence homology. We have characterized the basic structural and functional properties of VipF to confirm this original functional assignment. Sequence conservation analysis indicates two putative CoA-binding regions within VipF. Homology modeling and small angle X-ray scattering suggest a monomeric, dual-domain structure joined by a flexible linker. Each domain contains the characteristic beta-splay motif found in many acetyltransferases, suggesting that VipF may contain two active sites. Docking experiments suggest reasonable acetyl-CoA binding locations within each beta-splay motif. Broad substrate screening indicated that VipF is capable of acetylating chloramphenicol and both domains are catalytically active. Given that chloramphenicol is not known to be N-acetylated, this is a surprising finding suggesting that VipF is capable of O-acetyltransferase activity. Proteins 2016; 84:1422-1430. © 2016 Wiley Periodicals, Inc.

The Disulfide Bonds within BST-2 Enhance Tensile Strength during Viral Tethering.

Human BST-2/tetherin is a host factor that inhibits the release of enveloped viruses, including HIV-1, HIV-2, and SIV, from the cell surface by tethering viruses to the host cell membrane. BST-2 has an α-helical ectodomain that forms disulfide-linked dimers between two monomers forming a coiled coil. The ectodomain contains three cysteine residues that can participate in disulfide bond formation and are critical for viral tethering. The role of the disulfides in viral tethering is unknown but proposed to be for maintaining the dimer. We explored the role of the disulfides in the structure of BST-2 using experimental, biophysical methods. To understand the role of the disulfides in viral tethering, we used a new approach in viral tethering, steered molecular dynamics. We find that the disulfides coordinate the unfolding of the BST-2 monomers, which adds tensile strength to the coiled coil. Structural differences between oxidized and reduced BST-2 are apparent during unfolding, showing the monomers slide past each other in the absence of the disulfides. We found no evidence to support dissociation of the dimer upon reduction of the disulfide bonds. Moreover, the structure of BST-2 in the absence of the disulfides is similar to that of the oxidized form of BST-2, supporting previous X-ray crystallography and cellular work that showed the disulfides are not required for expression of BST-2. These data provide new insights into viral tethering by using novel techniques in the analysis of BST-2 to give amino acid level insight into functions of BST-2.