Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human replication protein A.

The RecQ helicases are involved in several aspects of DNA metabolism. Five members of the RecQ family have been found in humans, but only two of them have been carefully characterized, BLM and WRN. In this work, we describe the enzymatic characterization of RECQ1. The helicase has 3' to 5' polarity, cannot start the unwinding from a blunt-ended terminus, and needs a 3'-single-stranded DNA tail longer than 10 nucleotides to open the substrate. However, it was also able to unwind a blunt-ended duplex DNA with a "bubble" of 25 nucleotides in the middle, as previously observed for WRN and BLM. We show that only short DNA duplexes (30 bp) can be unwound by RECQ1 alone, but the addition of human replication protein A (hRPA) increases the processivity of the enzyme (100 bp). Our studies done with Escherichia coli single-strand binding protein (SSB) indicate that the helicase activity of RECQ1 is specifically stimulated by hRPA. This finding suggests that RECQ1 and hRPA may interact also in vivo and function together in DNA metabolism. Comparison of the present results with previous studies on WRN and BLM provides novel insight into the role of the N- and C-terminal domains of these helicases in determining their substrate specificity and in their interaction with hRPA

Molecular probes and switches for functional analysis of receptors, ion channels and synaptic networks

Photochromic switches and genetically encoded biosensors have become powerful tools for monitoring and modulating the activity of neurons and neuronal networks. Our understanding of the mechanisms underlying the development and functioning of the nervous system has greatly advanced in recent years thanks to advancements in these effective molecular and genetic tools. The idea of this Special Research Issue of Frontiers in Molecular Neuroscience is to provide an overview of the approaches in this area of research, and to present new applications in molecular imaging of ions and remote activation of receptors, ionic channels and synaptic networks. The issue contains experimental and methodological papers as well as review articles dealing with molecular tools for investigation and modulation of neuronal function. It can be divided into two main sections: (i) genetically encoded probes for non-invasive monitoring of ions and ATP; and (ii) optogenetic and optopharmacologic tools for control of neuronal activity with light

Two dimensional patterning of fluorescent proteins in hydrogels.

This work describes the successful micropatterning of hybrid systems consisting of hydrogel-dispersed optically active and controllable proteins on solid surfaces without degradation of the photophysical properties of the light-emitting biomolecules. It demonstrates the preservation of the luminescence properties of proteins entrapped into isolated microstructures of poly(acrylamide) gel. This way we can exploit both the structural and function-preserving properties of the hydrogels and the functionality of light-emitting proteins. We believe that this approach can open the way to the realization of nanopatterned optical memories based on photochromic biomolecules

New Red-Emitting Chloride-Sensitive Fluorescent Protein with Biological Uses

This work was financially supported by CTQ2017-85685-R, CTQ2017-85454-C2-1-P, and CTQ2017-86125-P (MICIU/AEI/ERDF). J.M.P. and J.D.V. thank UEQ for funding. J.V.P. is supported by an FPU fellowship (FPU17/04749). D.A. was supported by the Italian Cystic Fibrosis Foundation (grant FFC#3/2019) and by Fondazione Cassa Rurale Trento Rovereto (ref 2018.256). The authors acknowledge the Universidad of Granada (Spain) cell culture and microscopy central facilities (CIC-UGR).A new chloride-sensitive red fluorescent protein derived from Entacmaea quadricolor is described. We found that mBeRFP exhibited moderate sensitivity to chloride and, via sitedirected mutagenesis (S94V and R205Y), we increased the chloride affinity by more than an order of magnitude (kd = 106 ± 6 mM) at physiological pH. In addition, cis−trans isomerization of the chromophore produces a dual emission band with different chloride sensitivities, which allowed us to develop a ratiometric methodology to measure intracellular chloride concentrations.MICIU/AEI/ERDF CTQ2017-85685-R CTQ2017-85454-C2-1-P CTQ2017-86125-PUEQSpanish Government FPU17/04749Ministry of Health, ItalyItalian Cystic Fibrosis Research Foundation 3/2019Fondazione Cassa Rurale Trento Rovereto 2018.25

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Intracellular chloride ([Cl-](i)) andpH(pH(i)) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-](i) and pH(i), but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl-and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-](i) and pH(i) at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-](i) and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-](i) in response to hypercapnia and seizure activity.Peer reviewe