UVI31+ Is a DNA Endonuclease That Dynamically Localizes to Chloroplast Pyrenoids in C. reinhardtii

Abstract

UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.

A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

“…The protease activity of PI-PLC on the substrate protein UVI31+ [31], [32] was inhibited by the protease inhibitor leupeptin, while other inhibitors like AEBSF were unstable during a long incubation (Fig. 2A).…”

“…Each reaction mixture (30 µL total volume) contained 13 µM purified UVI31+ protein [31], [32] (14 kDa) and 0.2 units PI-PLC in 50 mM ammonium bicarbonate, and was incubated overnight at 37°C. The protein was then denatured by the addition of 7 µL SDS-denaturing solution (200 mM Tris-HCl pH 6.8, 8% SDS (w/v), 40% glycerol (v/v), 4% 2-mercaptoethanol (w/v), 50 mM EDTA pH 8.0, and 0.08% bromophenol blue (w/v) and heating at 100°C for 3 min.…”

Monothiol Glutaredoxin–BolA Interactions: Redox Control of Arabidopsis thaliana BolA2 and SufE1

“…In S. pombe, E. coli, and C. reinhardtii, the expression of BolA homologs is induced by stress (Aldea et al, 1989;Lee et al, 1994;Shukla et al, 2012) and human BOLA1 prevents mitochondrial morphology aberrations induced by GSH depletion by reducing the associated oxidative shift of the thiol redox potential (Willems et al, 2013). These functions may be related to the DNA-binding activity of BolA proteins which could act as transcriptional regulators (Freire et al, 2009;Cheng et al, 2011b;Guinote et al, 2011) or endonucleases (Shukla et al, 2012). Interestingly, the DNAbinding activity of several transcription factors of bacteria and fungi is controlled by redox posttranslational modifications (D'Autreaux and Toledano, 2007).…”

“…Indeed, BolA was previously defined as a transcriptional regulator only based on structural evidence indicating the presence of a K-homology domain typical of nucleic acid-binding proteins (Kasai et al, 2004;Chin et al, 2005). This capacity was further addressed by showing that Ehrlichia chaffeensis and E. coli BolAs bind to some promoters of target genes and that an ortholog from Chlamydomonas reinhardtii, uvi31 + , exhibits DNA endonuclease activity (Cheng et al, 2011b;Guinote et al, 2011;Shukla et al, 2012). Although most bacteria have two BolA-like proteins, a single study addressed the function of E. coli yrbA/ibaG in acid stress resistance (Guinote et al, 2012).…”

Structural and Spectroscopic Insights into BolA-Glutaredoxin Complexes

“…The Helix-turn-helix (HTH) Motif of BolAs Is Potentially Involved in DNA Binding-Currently, only bacterial BolA_H members have been shown to act as transcriptional regulators by binding to gene promoters (34,35), whereas Uvi31ϩ, a BolA_H from Chlamydomonas reinhardtii, likely exhibits endonuclease activity (36). The capacity to bind nucleic acids is in line with the fact that BolA possesses an overall fold related to the one found in type II K-homology (KH) domain-containing proteins (33) known to bind nucleic acids.…”