Substrate specificity of HeLa endonuclease R. A G-specific mammalian endonuclease.
We examined the substrate specificity of endonuclease R (endo R) a mammalian endonuclease that cleaves G.C-rich DNA sequences. The best substrates for double-stranded cleavage were homopolymeric stretches of poly(dG).poly(dC). Plasmids which contain other G-rich sequences were also cleaved but at a reduced frequency. These included the telomeric sequences, d(G4T2) and d(G2-6A), which were cleaved at approximately one-third the frequency of d(G)n.d(C)n. The alternating copolymer d(GA) and the terminal sequences of adeno-associated virus d(G1-3T/A) were also cut. Poly(dA).poly(dT) and the alternating copolymer d(GC)n were not detectably cleaved. Although endo R has a nicking activity which converts supercoiled plasmids to nicked circular DNA, the nicking activity is random with respect to plasmid sequences. Specific cleavage of G-rich sequences appears to occur by a concerted double-stranded mechanism. The cleavage pattern within the G-rich runs suggests that cleavage can occur anywhere within the G-rich region. Product ligation experiments indicate that a limited number of cleavage events (1-2) occur/molecule. Inasmuch as the best substrates for endo R are d(G)n.d(C)n and telomeric sequences, we suggest that endo R may directly recognize and cleave DNA that contains G.G base pairing.
Gottlieb
J
J
Department of Microbiology, State University of New York, Stony Brook Medical School 11794.
Muzyczka
N
N
D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011067:Poly dA-dT / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D013379:Substrate Specificity
D004270:DNA, Circular; D004278:DNA, Superhelical; D011089:Polydeoxyribonucleotides; D011067:Poly dA-dT; C019530:poly(dC-dG); C017650:polydeoxy(guanine-thymine) nucleotide; D004247:DNA; D004706:Endodeoxyribonucleases; C064670:endonuclease R