Nayana A Patil1, Bhakti Basu2, Deepti D Deobagkar3, Shree K Apte4, Dileep N Deobagkar5. 1. Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India; Department of Zoology, Centre for Advanced Studies, Savitribai Phule Pune University, Pune 411007, India. 2. Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India. 3. Department of Zoology, Centre for Advanced Studies, Savitribai Phule Pune University, Pune 411007, India. 4. Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India. Electronic address: aptesk@barc.gov.in. 5. Department of Zoology, Centre for Advanced Studies, Savitribai Phule Pune University, Pune 411007, India. Electronic address: dileep_deobagkar@hotmail.com.
Abstract
BACKGROUND: Control of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans. METHODS: C-5 methyl cytosines (m5C) were detected by immuno-blotting with m5C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry. RESULTS: Methylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m4C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m5C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc. CONCLUSION: DR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m5C. GENERAL SIGNIFICANCE: Although, dispensable under optimal growth conditions, the presence of m5C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.
BACKGROUND: Control of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans. METHODS:C-5 methyl cytosines (m5C) were detected by immuno-blotting with m5C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry. RESULTS: Methylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m4C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m5C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc. CONCLUSION: DR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m5C. GENERAL SIGNIFICANCE: Although, dispensable under optimal growth conditions, the presence of m5C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.
Authors: Giovanni Gallo; Ioannis Mougiakos; Mauricio Bianco; Miriam Carbonaro; Andrea Carpentieri; Anna Illiano; Pietro Pucci; Simonetta Bartolucci; John van der Oost; Gabriella Fiorentino Journal: mBio Date: 2021-12-07 Impact factor: 7.867