| |  | | | | | | A physical map of human Alu repeats cleavage by restriction endonucleases | |
| | Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns of Alu repeats cleavage by restriction endonucleases. We have proposed a method of restriction analysis of Alu repeats sequences in silico.
Simple software to analyze Alu repeats database has been suggested and Alu repeats digestion patterns for several restriction enzymes' recognition sites have been constructed. Restriction maps of Alu repeats cleavage for corresponding restriction enzymes have been calculated and plotted. Theoretical data have been compared with experimental results on DNA hydrolysis with restriction enzymes, which we obtained earlier.
Alu repeats digestions provide the main contribution to the patterns of human chromosomal DNA cleavage. This corresponds to the experimental data on total human DNA hydrolysis with restriction enzymes.
| |
| |
| | | | | Substrate specificity of new methyl-directed DNA endonuclease GlaI | |
| | Recently, we have discovered site-specific endonucleases, which recognize and cleave only DNA sequences with 5-methylcytosine. Two specificities of such endonucleases have been described. Enzymes BisI, BlsI, and GluI are isoschizomers and hydrolyze the DNA sequence 5'-GCNGC-3'/3'-CGNCG-5', which is methylated in different ways. The enzyme GlaI cleaves the DNA sequence 5'-GCGC-3'/3'-CGCG-5' if there are two, three or four 5-methylcytosines. The goal of the present work is to study in detail the composition of recognition sequence and effect of the methylated cytosines on the efficiency of DNA cleavage by the methyl-directed DNA endonuclease GlaI. In a recent work we have studied the dependence of GlaI activity on the quantity and location of 5-methylcytosines in the enzyme recognition sequence 5'-GCGC-3'/3'-CGCG-5'. A significant DNA cleavage has been observed for oligonucleotide duplexes, which include either three or four 5-methylcytosines. In this work we have studied dependence of the GlaI activity on quantity and location of methylated cytosines, as well as on composition of the recognition sequence.The list of good substrates for GlaI includes a fully methylated site 5'-CGCG-3'/3'-GCGC-5', sites with three cytosines of a general structure 5'-PuMGM-3'/3'-PyGMG-5', and one recognition sequence with two methylated cytosines 5'-AMGT-3'/3'-TGMA-5', where M is 5-methylcytosine. GlaI intermediate substrates include sites with three methylated cytosines of a general structure 5'-GMPuM-3'/3'-MGPyG-5', as well as a site with two methylcytosines 5'-GMGT-3'/3'-CGMA-5'. The site 5'-GMGC-3'/3'-CGMG-5' may be considered a low activity substrate. | |
| |
| | | | | Dependence of site-specific endonuclease GlaI activity on quantity and location of methylcytosines in the recognition sequence 5’-GCGC-3’. | |
| | The activity dependence of site-specific endonuclease GlaI that recognizes and hydrolyzes only methylated DNA sequence 5’-GCGC-3’ on the quantity and location of 5-methylcytosines in enzyme’s recognition sequence has been studied. A significant DNA cleavage has been observed for oligonucleotides duplexes containing four and three 5-methylcytosines or two internal modified bases. The cleavage efficiency is maximal for DNA duplex with four 5-methylcytosine and decreases when a number of methylated bases are lower. GlaI hydrolyzes recognition sequences with 5-methylcytosines but not with N4-methylcytosines. | |
| |
| | | |
| | | | | |
|
