Endonuclease Enzymes

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. 

Bacterial cells produce many enzymes that act to degrade various forms of DNA. Of special interest are the restriction endonucleases, enzymes that recognize specific base sequences in double-stranded DNA and catalyze... 

Nelson, M. and M. McClelland 1992. Use of DNA methyl transfer ase/endonuclease enzyme combinations for megabase mapping of chromosomes. Meth. Enzymol. 216 279-303. 

Other than proteins, the immobilized enzyme was also used to digest DNA. For instance, the restriction endonuclease enzyme HaeJR was immobilized on an amine-modified PMMA surface to digest DNA [1063]. 

If recombinant DNA and related techniques in molecular biology are to be applied meaningfully to cestodes, functional nucleic acids have to be prepared such that the DNA can be cleaved by restriction endonuclease enzymes (which digest the double-stranded DNA at highly specific sites, each recognizing a precise nucleotide sequence) and the RNA can be used to... 

The discovery of restriction endonucleases was of crucial importance in sequencing DNA. These enzymes recognize a specific sequence of four to eight bases in double-stranded DNA and cleave the DNA at a precise point in this sequence. For example, the restriction endonuclease known as AIul cleaves the sequence AGCT between the G and C and the one known as Pstl cleaves the sequence CTGCAG between the A and the G. These restriction endonuclease enzymes provide a reproducible way to produce precisely defined fragments of an appropriate size for sequencing. 

I) PCR RFLP (Figure 1) Species-specific fragments are amplified and then cut with the allele-specific endonuclease enzyme Ita 1 (FnuH 4, GCN.GC). This method provides qualitative results and is suitable for diagnosis of the presence or absence of the mutation in single spore isolates [8]. It has been adapted for 5. graminis, Mycosphaerella fijiensis and V. inaequalis [13]. 

Add one ninth volume of the tenfold concentrated restriction endonuclease buffer provided by the manufacturer, and 80 U of EcoRl restriction endonuclease enzyme. 

Restriction Endonucleases - Enzymes that catalyze the double-strand cleavage of DNA at specific base sequences. 

Figure 3.6 Heterologous (foreign) DNA (from either genomic DNA or a cDNA library) (see Section 3.2) is cut with two different restriction endonuclease enzymes (Eco Rl Xho I) producing a restriction digest.

Digestion of DNA fragments with restriction endonuclease enzymes, fragment purification by electroelution from electrophoresis gels, ligation and transformation procedures have been described (15). DNA sequence determination, SI nuclease protection and primer extension experiments were done as described (7). 

Restriction endonuclease Enzymes that catalyze the double-strand cleavage of DNA at specific base sequences. Many restriction endonucleases with different sequence specificities have been found in bacteria they are used extensively in molecular genetics. 

Loureiro, 2000 Deak, 2002 Capece et al., 2003). Each requires initial digestion of the harvested DNA using restriction endonucleases, enzymes that cleave DNA at specific nucleotide sequences unique for that enzyme. The DNA digest is the amplified at specific or randomly selected regions by polymerase chain reaction (PCR). Fragments are subsequently separated electrophoretically and their patterns compared against those of other isolates or databases. 

EcoRV is a hydrolase enzyme that belongs to the nuclease enzymes (EC 3.1) and is responsible for cleaving the P-O bond of phosphodiester in DNA and RNA. Exonuclease enzymes hydrolyze the P-O phosphodiester bond of the terminal mononucleotide and are not sequence specific [31]. Endonuclease enzymes include both examples, that is, enzymes that are not sequence specific and those that are sequence specific (e.g., type II restriction endonucleases) [32]. Type II restriction endonucleases in bacteria, archaea, and algae use their sequence specificity to bind and cleave the foreign DNA of invading viruses. These enzymes are especially interesting, as they combine their remarkable sequence specificity with high catalytic rates of phosphodiester hydrolysis. 

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