| Deinococcus radiodurans,widely known as one of the toughest bacteria,has manifested exceptional resistance to DNA damaging agents,including a high dose of ionizing irradiation,desiccation,ultraviolet radiation,oxidative stress,and genotoxic chemicals.As an ideal model organism for studying DNA damage repair and genomic stability maintenance,investigation on the survival mechanism of D.radiodurans will be of great significance to understand the life process and unique laws under extreme environmental stress.In this research,we performed biochemical and molecular biological methods to map the genome-wide DNA methylation patterns and identified the relevant DNA methyltransferase?MTase?in D.radiodurans.N4-cytosine DNA methylation played a crucial role in maintaining the genome stability of D.radiodurans.The biochemical properties of the DNA methyltransferase were characterized as well.And the epigenetic regulation of DNA methylation on gene transcriptional level of D.radiodurans was studied using RNA sequencing.?1?The single-molecule,real-time sequencing?SMRT-Seq?was performed to re-sequence the genome of D.radiodurans R1?DraR1?and predicted the methylome of this organism.We demonstrated that N4-cytosine methylation is the epigenetic marker of DraR1 using MTase gene knockout,ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry?UHPLC-QQQ-MS/MS?combined with SMRT-Seq.The genomic DNA?gDNA?of this bacterium contains around 1.3‰molar ratio of 4mC/dC,0.4‰of 6mA/dA and an undetectable trace of5mC/dC,which suggests that N4-cytosine methylation is the major methylated residue of DraR1.5'-C4mCGCGG-3',a novel motif,was identified and was fully attributable to the presence of M.DraR1 MTase that could recognize this motif and methylate the2nd cytosine at the atom of N4.In addition,the other three DNA MTases,ORF2230P,ORF14075P,and ORF15636P,are contributing to 6mA modification,but no any motifs associated with this modification was identified.?2?The amino acid sequences of M.DraR1 contains a canonical N-terminal SAM-binding motif?‘FxGxG'?followed by a centrally-located target recognition domain?TRD?and a C-terminal catalytic motif?‘SPPY'?,which appears to be the typical?-class of DNA 4mC MTase.EMSA assay also confirmed that the purified M.DraR1protein binds strictly with DNA substrates containing‘CCGCGG'sequence in the presence of SAM.In vivo and in vitro restriction digestion pattern analysis served as another powerful evidence to confirm that M.DraR1 is able to protect those DNA substrates from digestion by restriction endonucleases that can recognize and cut the same sequence,just as SacII,a cognate restriction endonuclease known to be sensitive to cytosine modification.These results established the first explicit report of M.DraR1as an?-class N4-Cytosine MTase that methylates the 5'-CCGCGG-3'motifs in contrast to methylation of the 2nd cytosine at the position C5 of the motifs as is in most cases.?3?We also demonstrated that the biological functions of 4mC modification in DraR1 using phenotypic assays combined with RNA sequencing.Results showed that knock out of M.DraR1 gene does not significantly affect growth under optimal conditions,but exhibits an increased sensitivity to high dose of DNA damage factors,higher spontaneous mutation frequency,enhanced recombination and transformation efficiency,which suggesting that 4mC methylation might act as a methylation barrier against foreign DNA and therefore contributes to genome stability.Moreover,cells lacking 4mC modification were characterized by the chromosome dispersion and nucleoid enlargement.RNA-seq analysis indicated that the absence of 4mC led to the enrichment of transcripts involved in DNA damage response and repair,which suggests that 4mC modification may regulate the transcriptional level of DraR1 through affecting its DNA conformation and thus participate in maintaining the stability of its own genome.In conclusion,we characterized the DNA N4-cytosine methylation in the extremophile D.radiodurans and addressed its possible association with transcriptional expression patterns,providing new insights into the properties and roles of N4-cytosine DNA methylation in the maintenance of genomic integrity in this organism.To the best of our knowledge,this was the first work to reveal the epigenetic regulation of 4mC methylation in D.radiodurans.These results not only deepened the new understanding of N4-cytosine DNA methylation contributes to the maintenance of genomic stability in D.radiodurans,but also lays the foundation for further research on the mechanisms of epigenetic regulation by R-M systems in bacteria. |
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