Base pairs endonucleases

Endonuclease-catalyzed hydrolysis of DNA at the internucleosomal linker regions into multimers of 180 base pairs which are visualized by electrophoresis as a ladder of nuclear DNA fragments. Access of the endonuclease to DNA is facilitated by depletion of polyamines, and the activity of the enzyme is mcrea.sed by and decreased by ADP-tibosylation. Thus, agents that increase intracellular Ca " or inhibit l>oly(ADP-ribose) polymerase can induce apoptosi.s. ... 

Figure 36-22. Mismatch repair of DNA. This mechanism corrects a single mismatch base pair (eg, C to A rather than T to A) or a short region of unpaired DNA. The defective region is recognized by an endonuclease that makes a single-strand cut at an adjacent methylated GATC sequence. The DNA strand is removed through the mutation, replaced, and religated.

Mismatch Repair. Mispairs that break the normal base-pairing rules can arise spontaneously due to DNA biosynthetic errors, events associated with genetic recombination and the deamination of methylated cytosine (Modrich, 1987). With the latter, when cytosine deaminates to uracil, an endonuclease enzyme, /V-uracil-DNA glycosylase (Lindahl, 1979), excises the uracil residue before it can pair with adenine at the next replication. However, 5-methyl cytosine deaminates to form thymine and will not be excised by a glycosylase. As a result, thymine exits on one strand paired with guanine on the sister strand, that is, a mismatch. This will result in a spontaneous point mutation if left unrepaired. For this reason, methylated cytosines form spontaneous mutation hot-spots (Miller, 1985). The cell is able to repair mismatches by being able to distinguish between the DNA strand that exists before replication and a newly synthesized strand. 

Bacteria contain enzymes that catalyse the breaking of phosphodiester links between nucleotides in DNA at specific sites, to which the enzyme is directed by a short sequence of bases. These are known as restriction enzymes and they have resulted in remarkable progress in analysing sequences of DNA fragments. They are endonucleases, i.e., they cleave DNA at the phosphodiester bonds within, rather than at the ends, of DNA chains. They cleave bonds such that sequences of nucleotides, typically 4-8 base pairs, are produced. These are the restriction sequences. 

Enzymes, specialized proteins, are used as designing tools for genetic engineering. One of these enzyme tools consists of restriction endonucleases that recognize a specific series of base pairs. They split the DNA at these specific points. This splitting is called lysing , which in reality is simply the hydrolysis of DNA units as shown in the following structure ... 

To isolate a gene, scientists use surgical DNA scissors called restriction endonucleases (RE), proteins made by bacteria that cut DNA, based on specific rules. Each kind of RE—there are hundreds— recognizes specific sequences of 4-8 base pairs and cuts the DNA molecule at a specific spot (Figure 1.2). The biotechnologist selects... 

Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to four nucleotides of one strand unpaired at each resulting end. These unpaired strands are referred to as sticky ends (Fig. 9-3a), because they can base-pair with each other or with complementary sticky ends of other DNA fragments. Other restriction endonucleases cleave both strands of DNA at the opposing phosphodiester bonds, leaving no unpaired bases on the ends, often called blunt ends (Fig. 9-3b). 

Restriction endonucleases recognize short stretches of DNA (generally four or six base pairs) that contain specific nucleotide sequences. These sequences, which differ for each restriction endonuclease, are palindromes, that is, they exhibit two-fold rotational symmetry (Figure... 

The cuts in the two strands are made at the points indicated by the arrows. This one endonuclease will cut almost any DNA into long pieces averaging about 5000 base pairs each. These pieces can in turn be cleaved by other restriction endonucleases to form smaller fragments. Since there are about 2400 of these enzymes known, with 188 different specificities,536 it is possible to cut any piece of DNA down to a size of 100-500 base pairs, ideal for sequencing.537 539 Each fragment has known sequences at the two ends. Some restriction enzymes cleave outside their specific recognition sequence (see Table 26-2). Some recognize 16-nucleotide palindromes and cut at rare sites. 

The colicin El plasmid is a 4.43 MDa circular double stranded DNA molecule consisting of 6646 base pairs.1 Only one site is susceptible to cleavage by the restriction endonuclease ECoRl (Chapter 26) This feature has led to its widespread use in cloning of genes. 

The type II restriction endonucleases, which are the ones most widely used in molecular biology, are relatively small 50- to 100-kDa monomeric or dimeric proteins. About 2400 different enzymes with 188 different specificities had been isolated by 1995.83/84 The sites of attack, in most instances, are nucleotide sequences with a twofold axis of local symmetry.85 For example, the following sites of cleavage have been identified for two restriction endonucleases encoded by the DNA of R-factor plasmids of E. coli and for a restriction enzyme from Hemophilus influenzae. In the diagrams i are sites of cleavage, are sites of methyla-tion, and are local twofold axes (centers of palindromes) N can be any nucleotide with a proper base pairing partner. 

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