Structural and functional investigation of the DHH/DHHA1 family proteins in Deinococcus radiodurans

Abstract

DHH/DHHA1 family proteins have been proposed to play critical roles in bacterial resistance to environmental stresses. Members of the most radioresistant bacteria genus, Deinococcus, possess two DHH/DHHA1 family proteins, RecJ and RecJ-like. While the functions of Deinococcus radiodurans RecJ (DrRecJ) in DNA damage resistance have been well characterized, the role and biochemical activities of D. radiodurans RecJ-like (DrRecJ-like) remain unclear. Phenotypic and transcriptomic analyses suggest that, beyond DNA repair, DrRecJ is implicated in cell growth and division. Additionally, DrRecJ-like not only affects stress response, cell growth, and division but also correlates with the folding/stability of intracellular proteins, as well as the formation and stability of cell membranes/walls. DrRecJ-like exhibits a preferred catalytic activity towards short single-stranded RNA/DNA oligos and c-di-AMP. In contrast, DrRecJ shows no activity against RNA and c-di-AMP. Moreover, a crystal structure of DrRecJ-like, with Mg2+ bound in an open conformation at a resolution of 1.97 Å, has been resolved. Subsequent mutational analysis was conducted to pinpoint the crucial residues essential for metal cation and substrate binding, along with the dimerization state, necessary for DrRecJ-like's function. This finding could potentially extend to all NrnA-like proteins, considering their conserved amino acid sequence and comparable dimerization forms.

Graphical Abstract

Figure 1.

Domain arrangement and phenotypes of deletion mutant and complementation strains. (A) Schematic representation of the domain arrangements of DrRecJ-like and DrRecJ. (B) Growth curves of the wild-type strain R1 (WT), drrecJ single deletion mutant (△J), drrecJ-like single deletion mutant (△J-like), and the drrecJ-like/recJ double deletion mutant (△J/△J-like) at 30 and 37 degrees. (C) The WT, △J, △J-like, △J/△J-like and related complementation strains (△J + J and △J-like + J-like) were treated with 0.1% tween 20 (for 30 min), 50 mM H₂O₂ (for 30 min), FUV (for 400 J/m²), or 40 μg/ml MMC (for 30 min). Cells were diluted and spotted on plates. Plates were cultured for 2−3 days at 30°C or 37°C.

Figure 2.

Transcriptome analysis by RNA-seq. (A) Clustered heatmap showing differential gene expressions (DGEs) in WT, △J, △J-like, and △J/△J-like mutant strains. (B) Venn diagram analysis of up-regulated (left) and down-regulated (right) DEGs in △J versus WT strain (yellow), △J-like versus WT strain (cyan), and △J /△J-like mutants versus WT strain (pink). (C) Volcano plot analysis showing DEGs profile between △J-like and WT strain. The gray dashed line indicates |log₂ fold change| > 1 and adjusted P-value < 0.005. (D) The top20 KEGG enrichment pathways for DEGs in pairwise comparison of △J-like versus WT. The rich factor refers to the ratio of the number of DEGs in the pathway and the number of all annotated genes in the pathway.

Figure 3.

The enzymatic activity analysis of DrRecJ-like. The enzymatic activity analysis of DrRecJ-like. (A) Metal preference test. Hydrolysis of 100 nM 20 nt 5′FAM labeled RNA by DrRecJ-like (500 nM) in the presence of indicated divalent metal cations (10 mM). (B) Divalent metal ion concentrations test. 100 nM 20 nt 5′FAM labeled ssRNA was incubated with 100 nM DrRecJ-like (when MnCl₂ or CoCl₂ was used), or 200 nM DrRecJ-like (when NiCl₂ was used), or 500 nM DrRecJ-like (when MgCl₂ was used). Different concentrations (0, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mM) of divalent metal cations were introduced. (C) Digestion comparison between ssRNA and ssDNA by DrRecJ-like. 100 nM 20 nt 3′FAM labeled ssRNA or ssDNA was incubated with different concentrations of DrRecJ-like (0, 6.25, 12.5, 25, 50, 100, 200 nM) and 1 mM Mn²⁺. (D) Optimum substrate lengths determination. 0.8 mM substrates with different length (2, 3, 4, 5 nt) were incubated with 0.5 μM DrRecJ-like and 1 mM Mn²⁺ for various durations (0, 5, 10, 15, 20, 30 min), and the reaction products were resolved by TLC. M represents AMP standard. (E) Comparison of the cleavage efficiency of DrRecJ-like on different substrates based on the TLC results. The reaction conditions are the same as that in figure 3D and Supplementary Figure S3A. Three technical replicates were conducted. (F) Digestion assays of c-di-AMP. 0.8 mM c-di-AMP was incubated with 2 μM DrRecJ-like and 1 mM Mn²⁺ for various durations (0, 5, 10, 15, 20, 30 min), and the reaction products were resolved by TLC. M1 represents AMP standard, and M2 represents pApA standard. (G) The inorganic phosphate assays testing the digestion of pAp, pApA and ApAp. 200 μM pAp, pApA, or ApAp was incubated with 200 μM DrRecJ-like (or DrRecJ-like^D95A, or DrRecJ) or 2 μM DrCysQ, and 1 mM Mn²⁺ for 1 h. The release of Pi was detected at OD₆₆₀, by an inorganic phosphate assay kit. 200 μM Pi standard was set as a control. Three technical replicates were conducted. (H) A model for catalysis of ApAp by DrRecJ-like. Red arrows indicate the cleavage sites, and the blue arrow indicate the digestion direction. (I) The levels of in vivo oligonucleotide content were measured in the wild-type and DrRecJ-like deletion strain. The concentrations of oligonucleotides were calculated per milliliter of culture (OD₆₀₀= 1). Three technical replicates were conducted. ‘*’ indicated P< 0.05.

Figure 4.

The crystal structure and key residues for catalysis and dimerization of DrRecJ-like. (A) The overall structure of DrRecJ-like dimer. One protomer of the dimer is shown in color (DHH domain, violet; DHHA1, marine) and the other protomer of the dimer is shown in white. (B) Zoom-in view of the catalytic core. The key residues involved in metal ion coordination and substrate interaction are shown as sticks and colored orange and cyan, respectively. (C) and (D) Denaturing PAGE gel showing the reduced nuclease activity of point-mutated DrRecJ-like proteins (alanine substitutions of key residues involved in metal ion coordination or substrates binding). 5′FAM-labeled 20 nt ssDNA (200 nM) was incubated with different concentrations of DrRecJ-like proteins (0, 0.1 and 0.5 μM) in the presence of 1 mM Mn²⁺ (see methods). (E) Zoom-in view of the dimerization interface. Key residues for dimerization are shown as sticks and labeled. (F) Sequence alignment of the dimerization areas of the structured NrnA homologs. Dra, D. radiodurans RecJ-like (PDB ID: 8IOO); Sa1, S. aureus SA0013 NrnA (PDB ID: 8IU7); Sa2, S. aureus SA0013 (GdpP) (PDB ID: 5XSI); Bsu, B. subtilis YtqI (PDB ID: 5J21); Mtu, Mycobacterium tuberculosis Rv2837c (CnpB) (PDB ID: 5JJU); Tma, T. maritima TM1595 (PDB ID: 5O1U); Msm, Mycobacterium smegmatis MC2 155 NrnA (PDB ID: 4LS9); Sha, Staphylococcus haemolyticus JCSC1435 NrnA (PDB ID: 3DEV); Bfr, Bacteroides fragilis YCH46 (PDB ID: 3W5W). Secondary structural elements are depicted according to the structure of the DrRecJ-like protein characterized in this study and displayed at the top of sequences. Putative residues involved in protein dimerization are boxed in light green.

Figure 5.

Functional analysis of the dimerization of DrRecJ-like. (A) Gel filtration confirmation of the DrRecJ-like dimerization-defective mutant. A set of protein standards with known molecular masses, including aprotinin (6.5 kDa), RNase A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa), and conalbumin (75 kDa), were used to calibrate the Superdex 75 10/300GL. DrRecJ-like^wt eluted around 10 ml, indicating a dimeric form. DrRecJ-like^m eluted around 11 ml, indicating a monomeric form. (B) Comparison of the digestion activities of DrRecJ-like^wt and DrRecJ-like^m towards oligonucleotides. 200 nM 5′FAM-labeled 20 nt ssDNA or ssRNA was incubated with different concentrations of DrRecJ-like proteins (0, 0.1, 0.2, 0.4 and 0.8 μM) in the presence of 1 mM Mn²⁺ (see methods). (C) The WT, △J-like, and △J-like strains complemented with DrRecJ-like^wt or DrRecJ-like^m (△J-like + J-like or △J-like + J-like^m) were treated with 0.1% tween 20 (for 30 min), 50 mM H₂O₂ (for 30 min), FUV (for 400 J/m²), or 40 μg/ml MMC (for 30 min). Cells were diluted and spotted on plates. Plates were cultured for 2−3 days at 30°C.