Characterisation of sequence-structure-function space in sensor-effector integrators of phytochrome-regulated diguanylate cyclases.

Understanding the relationship between protein sequence, structure and function is one of the fundamental challenges in biochemistry. A direct correlation, however, is often not trivial since protein dynamics also play an important functional role-especially in signal transduction processes. In a subfamily of bacterial light sensors, phytochrome-activated diguanylate cyclases (PadCs), a characteristic coiled-coil linker element connects photoreceptor and output module, playing an essential role in signal integration. Combining phylogenetic analyses with biochemical characterisations, we were able to show that length and composition of this linker determine sensor-effector function and as such are under considerable evolutionary pressure. The linker length, together with the upstream PHY-specific domain, influences the dynamic range of effector activation and can even cause light-induced enzyme inhibition. We demonstrate phylogenetic clustering according to linker length, and the development of new linker lengths as well as new protein function within linker families. The biochemical characterisation of PadC homologs revealed that the functional coupling of PHY dimer interface and linker element defines signal integration and regulation of output functionality. A small subfamily of PadCs, characterised by a linker length breaking the coiled-coil pattern, shows a markedly different behaviour from other homologs. The effect of the central helical spine on PadC function highlights its essential role in signal integration as well as direct regulation of diguanylate cyclase activity. Appreciation of sensor-effector linkers as integrator elements and their coevolution with sensory modules is a further step towards the use of functionally diverse homologs as building blocks for rationally designed optogenetic tools.

Putative Origins of Cell-Free DNA in Humans: A Review of Active and Passive Nucleic Acid Release Mechanisms.

Through various pathways of cell death, degradation, and regulated extrusion, partial or complete genomes of various origins (e.g., host cells, fetal cells, and infiltrating viruses and microbes) are continuously shed into human body fluids in the form of segmented cell-free DNA (cfDNA) molecules. While the genetic complexity of total cfDNA is vast, the development of progressively efficient extraction, high-throughput sequencing, characterization via bioinformatics procedures, and detection have resulted in increasingly accurate partitioning and profiling of cfDNA subtypes. Not surprisingly, cfDNA analysis is emerging as a powerful clinical tool in many branches of medicine. In addition, the low invasiveness of longitudinal cfDNA sampling provides unprecedented access to study temporal genomic changes in a variety of contexts. However, the genetic diversity of cfDNA is also a great source of ambiguity and poses significant experimental and analytical challenges. For example, the cfDNA population in the bloodstream is heterogeneous and also fluctuates dynamically, differs between individuals, and exhibits numerous overlapping features despite often originating from different sources and processes. Therefore, a deeper understanding of the determining variables that impact the properties of cfDNA is crucial, however, thus far, is largely lacking. In this work we review recent and historical research on active vs. passive release mechanisms and estimate the significance and extent of their contribution to the composition of cfDNA.