mode of action of restriction enzymes.pptx

basic info about the topic

1. Restriction Enzymes

2. What are Restriction Enzymes ? Proteins that cut DNA. Restrict host range for certain bacteriophages

3. Restriction enzyme A restriction enzyme or restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites known as restriction sites.

4. Scan the DNA sequence Find a very specific set of nucleotides Make a specific cut

5. Molecular Scissors for Cutting DNA Precisely

6. History The term restriction enzyme originated from the studies of phage λ , a virus that infects bacteria and the phenomenon of host-controlled restriction and modification of such bacterial phage or bacteriophage. The phenomenon was first identified in work done in the laboratories of Salvador Luria and Giuseppe Bertani in the early 1950s.

7. Cont. The restriction enzymes studied by Arber and Meselson were type I restriction enzymes, which cleave DNA randomly away from the recognition site. In 1970, Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated and characterized the first type II restriction enzyme.

8. Occurrence These enzymes are found in bacteria and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction . Host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.

9. Bacteria produce restriction enzymes to protect against invading viral DNA/RNA.

10. The enzymes cut the invading DNA/RNA, rendering it harmless.

11. 3)Bacterium modifies its own restriction sites by methylation so enzyme cannot cut it

12. Restriction Modification System Restriction enzymes are paired with methylases . Restriction enzyme destroys unprotected = non-self DNA Methylase recognizes same sequence in host DNA and protects it by methylating it

13. Therefore, the restriction enzyme doesn’t destroy its own DNA. However the restriction enzyme can destroy foreign DNA

14. How Do Restriction Enzymes Work? Like all enzymes, restriction enzymes are highly specific. They cut DNA only within very precise recognition sequences. The red line shows where the enzymes will cut the DNA. Notice that all of these recognition sites are symmetrical, or what is called "palindromic." This means that the recognition sequence on one DNA strand reads in the opposite direction on the complementary strand .

15. Recognition site Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences can also be classified by the number of bases in its recognition site, usually between 4 and 8 bases, and the amount of bases in the sequence will determine how often the site will appear by chance in any given genome.

16. TYPES OF CUTS

17. blunt end sticky end

18. Blunt ends Restriction endonucleases can cut double-stranded DNA in a few different ways. Sometimes it cuts both strands at the same position, which causes  blunt ends.  

19. Sticky ends R.E cuts each strand at a different point causing overhangs to occur. An overhang means that one strand is longer than the other, and also referred as having sticky ends. 

20. Types Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV) based on their composition, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence.

21. Cont .. 1)Type I enzymes cleave at sites remote from a recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional protein with both restriction and methylase activities. 2)Type II enzymes cleave within or at short specific distances from a recognition site; most require magnesium for its functioning.

22. Cont.. 3 ) Type III enzymes cleave at sites a short distance from a recognition site; require ATP ,S- adenosyl -L-methionine stimulates the reaction but is not required; exist as part of a complex with a modification methylase . 4) Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA .

23. Applications Isolated restriction enzymes are used to manipulate DNA for different scientific applications They are used to assist insertion of genes into plasmid vectors during gene cloning and protein production experiments. Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms ( SNPs).

24. The different lengths of DNA generated by restriction digest also produce a specific pattern of bands after gel electrophoresis, and can be used for DNA fingerprinting. Restriction enzymes are used to digest genomic DNA for gene analysis by Southern blot.

25. Examples: Enzymes Source Recognition sequence Cut Eco RI Escherichia coli 5'GAATTC 3'CTTAAG 5'---G AATTC---3' 3 '---CTTAA G---5' Hin d III Haemophilus influenzae 5'AAGCTT 3'TTCGAA 5'---A AGCTT---3' 3 '---TTCGA A---5' Taq I Thermus aquaticus 5'TCGA 3'AGCT 5'---T CGA--- 3‘ 3'---AGC T---5' Sma I * Serratia marcescens 5'CCCGGG 3'GGGCCC 5'---CCC GGG- -3 ' 3'---GGG CCC---5'