Involvement of Rev1 in alkylating agent-induced loss of heterozygosity in Oryzias latipes.

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.

RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility.

Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmA(II) enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmA(II), rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmA(II) in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmA(II) activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmA(II), thereby facilitating TEL binding to the ribosome.

Methylation of 23S rRNA nucleotide G748 by RlmAII methyltransferase renders Streptococcus pneumoniae telithromycin susceptible.

Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmA(II), which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 µg/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 µg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmA(II) to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmA(II) renders S. pneumoniae TEL susceptible.

Staphylococcus Agr virulence is critical for epidermal colonization and associates with atopic dermatitis development.

Atopic dermatitis (AD) is commonly associated with colonization by Staphylococcus aureus in the affected skin. To understand the role of S. aureus in the development of AD, we performed whole-genome sequencing of S. aureus strains isolated from the cheek skin of 268 Japanese infants 1 and 6 months after birth. About 45% of infants were colonized with S. aureus at 1 month regardless of AD outcome. In contrast, skin colonization by S. aureus at 6 months of age increased the risk of developing AD. Acquisition of dysfunctional mutations in the S. aureus Agr quorum-sensing (QS) system was primarily observed in strains from 6-month-old infants who did not develop AD. Expression of a functional Agr system in S. aureus was required for epidermal colonization and the induction of AD-like inflammation in mice. Thus, retention of functional S. aureus agr virulence during infancy is associated with pathogen skin colonization and the development of AD.