Substrate specificity of new methyl-directed DNA endonuclease GlaI

Abstract

Background: 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 RESULTS: 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.

Conclusion: 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.

Figure 1

Cleavage of the oligonucleotide duplex G7*/G8 by endonucleases. Runs: 1 – DNA duplex without the enzyme; 2 – hydrolysis with restriction endonuclease Rsr2I; 3 – hydrolysis with restriction endonuclease AclI; 4, 5 – hydrolysis with endonuclease GlaI (30 units and 100 units, respectively).

Figure 2

Dependence of the hydrolyzed product (in %) (axis Y) on the time of reaction: G7*/G8 (5'-TTTTCGMGMTGACG-3'/3'-AAAAGMGMGACTGC-5') – ■, G8*/G7 (5'-CGTCAGMGMGAAAA-3'/3'-GCAGTMGMGCTTTT-5') – □, G3*/G8 (5'-TTTTCGMGCTGACG-3'/3'-AAAAGMGMGACTGC-5') – ▽, G8*/G3 (5'-CGTCAGMGMGAAAA-3'/3'-GCAGTCGMGCTTTT-5') – ▼, G4*/G7 (5'-CGTCAGMGCGAAAA-3'/3'-GCAGTMGMGCTTTT-5') – △, G7*/G4 (5'-TTTTCGMGMTGACG-3'/3'-AAAAGCGMGACTGC-5') – ▲, G11*/G12 (5'-TTTTCAMGMTGACG-3'/3'-AAAAGTGMGACTGC-5') – ●, G12*/G11 (5'-CGTCAGMGTGAAAA-3'/3'-GCAGTMGMACTTTT-5') – ○, G7*/G6 (5'-TTTTCGMGMTGACG-3'/3'-AAAAGMGCGACTGC-5') – .

Figure 3

Dependence of the hydrolyzed product (in %) (axis Y) on the time of reaction: a) G7*/G6 (5'-TTTTCGMGMTGACG-3'/3'-AAAAGMGCGACTGC-5') – ■, G6*/G7 (5'-CGTCAGCGMGAAAA-3'/3'-GCAGTMGMGCTTTT-5') – □, G5*/G8 (5'-TTTTCGCGMTGACG-3'/3'-AAAAGMGMGACTGC-5') – ●, G8*/G5 (5'-CGTCAGMGMGAAAA-3'/3'-GCAGTMGCGCTTTT-5') – ○, G13*/G14 (5'-TTTTCGTGMTGACG-3'/3'-AAAAGMAMGACTGC-5') – ▲, G14*/G13 (5'-CGTCAGMAMGAAAA-3'/3'-GCAGTMGTGCTTTT-5') – △, G4*/G7 (5'-CGTCAGMGCGAAAA-3'/3'-GCAGTMGMGCTTTT-5') – ; b) G13*/G14 (5'-TTTTCGTGMTGACG-3'/3'-AAAAGMAMGACTGC-5') – ▲, G14*/G13 (5'-CGTCAGMAMGAAAA-3'/3'-GCAGTMGTGCTTTT-5') – △, G20*/G21 (5'-TTTTCGMAMTGACG-3'/3'-AAAAGMGTGACTGC-5') – ▼, G21*/G20 (5'-CGTCAGTGMGAAAA-3'/3'-GCAGTMAMGCTTTT-5') – ▽.

Figure 4

Dependence of the hydrolyzed product (in %) (axis Y) on the time of reaction: a) G3*/G4 (5'-TTTTCGMGCTGACG-3'/3'-AAAAGCGMGACTGC-5') – ■, G4*/G3 (5'-CGTCAGMGCGAAAA-3'/3'-GCAGTCGMGCTTTT-5') – □, G15*/G16 (5'-TTTTCAMGTTGACG-3'/3'-AAAAGTGMAACTGC-5') – ●, G16*/G15 (5'-CGTCAAMGTGAAAA-3'/3'-GCAGTTGMACTTTT-5') – ○, G18*/G19 (5'-TTTTCGMGTTGACG-3'/3'-AAAAGCGMAACTGC-5') – ▲, G19*/G18 (5'-CGTCAAMGCGAAAA-3'/3'-GCAGTTGMGCTTTT-5') – △, G7*/G6 (5'-TTTTCGMGMTGACG-3'/3'-AAAAGMGCGACTGC-5') – ; b) G12*/G17 (5'-CGTCAGMGTGAAAA-3'/3'-GCAGTCGMACTTTT-5') – ▼, G17*/G12 (5'-TTTTCAMGCTGACG-3'/3'-AAAAGTGMGACTGC-5') – ▽, G18*/G19 (5'-TTTTCGMGTTGACG-3'/3'-AAAAGCGMAACTGC-5') – ▲, G19*/G18 (5'-CGTCAAMGCGAAAA-3'/3'-GCAGTTGMGCTTTT-5') – △.

Figure 5

GlaI activity on methylated oligonucleotide duplexes.