Cleavage by restriction endonucleases

B 10. Which of the following base sequences are probably not recognition sites for cleavage by restriction endonucleases Why not ... 

In prokaryotic DNA the major methylated bases are N -methyladenine (mA) and to a lesser extent Nl-methylcytosine. Methylation in bacteria occurs at specific sites. In E. coli, methylation of A residues in the sequence 5 -GATC-3 is involved in mismatch error correction, and it plays a role in controlling initiation of DNA replication. Methylation at other sites protects DNA against cleavage by restriction endonucleases (described here). Structural studies on a bacterial DNA methylase have shown that the bases undergoing methylation rotate completely out of the DNA duplex and into a catalytic pocket within the enzyme structure. Other enzymes that work on bases, such as uracil-N-glycosylase, operate similarly. 

N -Methyladenine is a methylated base found in prokaryotic DNA. In E. coli, methylation of A residues in the sequence 5 -GATC-3 is involved in mismatch error correction, and it plays a role in controlling initiation of DNA replication. Methylation at other sites protects DNA against cleavage by restriction endonucleases (described here). 

The detection of restriction fi agment length polymorphisms (RFLPs) facilitates prenatal detection of hereditary disorders such as sickle cell trait, beta-thalassemia, infant phenylketonuria, and Huntington s disease. Detection of RFLPs involves cleavage of double-stranded DNA by restriction endonucleases, which can detect subtle alterations in DNA that affect their recognized sites. Chapter 40 provides further details concerning the use of PCR and restriction enzymes for diagnosis. 

SPECIFICITY. Restriction endonucleases are highly specific in hydrolyzing doubled-stranded DNA. Not only do they recognize a specific sequence of four to six base pairs (up to 11 base pairs in case of Bgl I see Table II) but the base pairs must be patind/iomic in the two strands of DNA. The cleavage sites possess twofold rotational symmetry as shown in Scheme I for restriction endonuclease Aat II from AcdtobacXdA OUddtL (Table II A, T, G, C indicate deoxyribonucleotides in this paper). In this example, the T-C phosphodiester bond in each strand two residues distal to the symmetry axis is cleaved by restriction endonuclease Aat II. 

Fig. 2.12. Cleavage of DNA by restriction endonuclease EcoK I and joining by DNA ligase. The two shades of the DNA backbones represent DNA ends generated in different cleavage events.

FIGURE 7.4 In RARE cleavage, specific restriction endonuclease sites are protected with a complementary oligonucleotide. In the presence of RecA, a triplex DNA complex is formed that prevents methylation at the protected site(s), whereas unprotected sites are methylated by the corresponding methylase. After dissociation of the complex, the protected sites can be cleaved with the specific restriction endonuclease. 

Figure 7. Cleavage of cis- and trans-DDP modified SV40 DNA by restriction endonucleases that cut at a single unique site, (Reproduced with permission from Ref. 16. Copyright 1982, Academic Press.)...

Danna, K. and Nathans, D. (1971) Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Scl U.S.A., 68, 2913 -2917. 

Fig. 1 Nucleic acid samples available for electrochemical experiments, (a, b) naturally occurring DNAs (bl) dsDNA fragments of defined lengths and nucleotide sequences can be conveniently prepared by cleavage with restriction endonucleases (c) NAs (both DNA and RNA) synthesized by enzymes (d) fully synthetic DNAs and RNAs of limited lengths (e) PCR, can amplify the desired DNA segment from template DNA. ds, double-stranded ss, single-stranded, kbp, kilobase pairs. Covalently closed circles of sc, supercoiled and rel, relaxed DNA. oc, open circular DNA (containing at least one interruption of the sugar-phosphate backbone) lin, linear DNA. See text for more details.

Mattei, D. M., Goldberg, S., Morel, C., Azevedo, H.P., and Roitman, I. 1977. Biochemical strain characterization of Trypanosoma cruzi by restriction endonuclease cleavage of kinetoplast DNA. Federation of European Biochemical Societies Letters. 74 264-268. 

Fig. 24.3 The construction of a chimeric (or recombinant) DNA molecule hy joining together two DNA fragments produced by cleavage of different parental DNA molecules with the same restriction endonuclease.

This activity is intended to be performed in conjunction with Experiment 66. Restriction endonucleases, or restriction enzymes, cleave DNA at specific base sequences, fragmenting the DNA into smaller pieces. The two strands of a DNA double helix are cleaved at different places, resulting in uneven fragments called sticky ends. Cleavage of DNA by restriction enzymes is a required first step in various types of DNA analysis, including DNA fingerprinting and recombinant DNA technology. 

Fig. 1. Nucleotide sequence of the SFV 26 S RNA (top row), the corresponding amino acid sequence (middle row), and the amino acid sequence of the Sindbis virus structural proteins (bottom row). Nucleotides are numbered from the 5 end of the RNA molecule and all amino adds from the amino terminus of each protein. The amino- and the carboxyl-terminal ends of each protein are indicated hy arrows, glycosylation sites by triangles, and membrane-spanning regions of the viral glycoproteins by underlines for Sindbis virus and overlines for SFV. Amino acids in boxes are negatively charged (Asp and Glu), and those circled are positively charged (Lys and Arg). Some restriction endonuclease cleavage sites are shown on the nucleotide sequence. The alignment of the amino acid...

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