Dam methylase from Escherichia coli: kinetic studies using modified DNA oligomers: hemimethylated substrates.
AUTOR(ES)
Marzabal, S
RESUMO
We have measured steady-state kinetics of the N6-adenine methyltransferase Dam Mtase using as substrates non-selfcomplementary tetradecamer duplexs (d[GCCGGATCTAGACG]-d[CGTCTAGATCC-GGC]) containing the hemimethylated GATC target sequence in one or the other strand and modifications in the GATC target sequence of the complementary strands. Modifications included substitution of guanine by hypoxanthine (I), thymine by uracil (U) or 5-ethyl-uracil (E) and adenine by 2,6-diamino-purine (D). Thermodynamic parameters were obtained from the concentration dependence of the melting temperature (Tm) of the duplexes. Large differences in DNA methylation of duplexes containing single dI for dG substitution of the Dam recognition site were observed compared with the canonical substrate, if the substitution involved the top strand (on the G.C rich side). Substitution in either strand by uracil (dU) or 5-ethyluracil (dE) resulted in small perturbation of the methylation patterns. When 2,6-diamino-purine (dD) replaced the adenine to be methylated, small, but significant methylation was observed. The kinetic parameters of the methylation reaction were compared with the thermodynamic free energies and significant correlation was observed.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=307261Documentos Relacionados
- Retron for the 67-base multicopy single-stranded DNA from Escherichia coli: a potential transposable element encoding both reverse transcriptase and Dam methylase functions.
- The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.
- Contrasting effects of single stranded DNA binding protein on the activity of uracil DNA glycosylase from Escherichia coli towards different DNA substrates.
- Escherichia coli K-12 clones that overproduce dam methylase are hypermutable.
- Mechanistic studies of glutamine synthetase from Escherichia coli: kinetic evidence for two reaction intermediates in biosynthetic reaction.