Recognition of individual Escherichia coli transfer ribonucleic acids by 1-adenine-specific methyltransferase from rat liver.
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ABSTRACT:
SUBMITTER: Kraus J
PROVIDER: S-EPMC1184215 | biostudies-other | 1978 Jan
REPOSITORIES: biostudies-other
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Project description:The structure of the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase in complex with cognate DNA was determined at 1.89 A resolution in the presence of S-adenosyl-L-homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3') half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E.coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S-adenosyl-L-methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.
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Project description:We have investigated the structural basis of processive GATC methylation by the Escherichia coli DNA adenine methyltransferase, which is critical in chromosome replication and mismatch repair. We determined the contribution of the orthologically conserved phosphate interactions involving residues Arg(95), Asn(126), Asn(132), Arg(116), and Lys(139), which directly contact the DNA outside the cognate recognition site (GATC) to processive catalysis, and that of residue Arg(137), which is not conserved and contacts the DNA backbone within the GATC sequence. Alanine substitutions at the conserved positions have large impacts on processivity yet do not impact k(cat)/K(m)(DNA) or DNA affinity (K(D)(DNA)). However, these mutants cause large preferences for GATC sites varying in flanking sequences when considering the pre-steady state efficiency constant k(chem)/K(D)(DNA). These changes occur mainly at the level of the methylation rate constant, which results in the observed decreases in processive catalysis. Thus, processivity and catalytic efficiency (k(cat)/K(m)(DNA)) are uncoupled in these mutants. These results reveal that the binding energy involved in DNA recognition contributes to the assembly of the active site rather than tight binding. Furthermore, the conserved residues (Arg(95), Asn(126), Asn(132), and Arg(116)) repress the modulation of the response of the enzyme to flanking sequence effects. Processivity impacted mutants do not show substrate-induced dimerization as is observed for the wild type enzyme. This study describes the structural means by which an enzyme that does not completely enclose its substrate has evolved to achieve processive catalysis, and how interactions with DNA flanking the recognition site alter this processivity.
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Project description:DNA sequence analysis revealed that the putative yhdJ DNA methyltransferase gene of Escherichia coli is 55% identical to the Nostoc sp. strain PCC7120 gene encoding DNA methyltransferase AvaIII, which methylates adenine in the recognition sequence, ATGCAT. The yhdJ gene was cloned, and the enzyme was overexpressed and purified. Methylation and restriction analysis showed that the DNA methyltransferase methylates the first adenine in the sequence ATGCAT. This DNA methylation was found to be regulated during the cell cycle, and the DNA adenine methyltransferase was designated M.EcoKCcrM (for "cell cycle-regulated methyltransferase"). The CcrM DNA adenine methyltransferase is required for viability in E. coli, as a strain lacking a functional genomic copy of ccrM can be isolated only in the presence of an additional copy of ccrM supplied in trans. The cells of such a knockout strain stopped growing when expression of the inducible plasmid ccrM gene was shut off. Overexpression of M.EcoKCcrM slowed bacterial growth, and the ATGCAT sites became fully methylated throughout the cell cycle; a high proportion of cells with an anomalous size distribution and DNA content was found in this population. Thus, the temporal control of this methyltransferase may contribute to accurate cell cycle control of cell division and cellular morphology. Homologs of M.EcoKCcrM are present in other bacteria belonging to the gamma subdivision of the class Proteobacteria, suggesting that methylation at ATGCAT sites may have similar functions in other members of this group.
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