Dr.

Adel Abuzenadah

DNA Manipulative Enzymes

1- Nuclease Enzymes that cut,shorten or degrade nucleic acid molecules. A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids.

Examples of Restriction Enzymes

Target sequence Enzyme Organism from which derived (cut at *) 5' -->3' Ava I Bgl II Eco RI Anabaena variabilis Bacillus globigii Escherichia coli RY 13 C* C/T C G A/G G G* G A T C C A* G A T C T G* A A T T C * C C A/T G G GG*CC GCG*C A* A G C T T GTT*AAC GGTAC*C *G A T C *G A T C CTGCA*G CCC*GGG GAGCT*C G*TCGAC T*CGA C*CCGGG Bam HI Bacillus amyloliquefaciens

Eco RII Escherichia coli R245 Hae III Haemophilus aegyptius Hha I Hpa I Kpn I Mbo I Mbo I Pst I Sma I SstI Sal I Taq I Xma I Haemophilus haemolyticus Haemophilus parainflenzae Klebsiella pneumoniae Moraxella bovis Moraxella bovis Providencia stuartii Serratia marcescens Streptomyces stanford Streptomyces albus G Thermophilus aquaticus Xanthamonas malvacearum Hind III Haemophilus inflenzae Rd

Note: Only one strand of the target DNA is shown, in the interests of clarity. It will be noted that the omitted strand has the same sequence as the one show, but the nucleotides occur in the reverse order. Thus for example The complementary sequence for Sal I (with its point of cleavage indicated as above) is 5' -C A G C T * G-3'. These are hence said to

Dr. Adel Abuzenadah be palindromic sequences, and double-stranded cuts produce short sinlge-stranded cohesive ends. Exonucleases are enzymes that cleave nucleotides one at a time from an end of a polynucleotide chain. These enzymes hydrolyze phosphodiester bonds from either the 3' or 5' terminus of polynucleotide molecules. Examples - Exonuclease III. Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain that connects the RNA and DNA tracts. Restriction endonucleases (Restriction Enzymes) cleave DNA at specific sites, and are divided into three categories, Type I, Type II, and Type III, according to their mechanism of action. These enzymes are often used in genetic engineering to make recombinant DNA for introduction into bacterial, plant, or animal cells. 2- Ligase: join nucleic acid molecules together. In biochemistry, a ligase (from the Latin verb ligre "to bind" or "to glue together") is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group pendant to one of the larger molecules.

3- DNA polymerase: Make copies of molecules. A DNA polymerase is an enzyme that assists in DNA replication. Such enzymes catalyze the polymerization of deoxyribonucleotides alongside a DNA strand, which they "read" and use as a template. The newly-polymerized molecule is complementary to the template strand and identical to the template's partner strand. All DNA polymerases synthesize DNA in the 5' to 3' direction. No known DNA polymerase is able to begin a new chain (de novo). They can only add a nucleotide onto a preexisting 3'-OH group. For this reason, DNA polymerase needs a primer at which it can

Dr. Adel Abuzenadah add the first nucleotide. Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by another enzyme called primase. An enzyme known as a helicase is required to unwind DNA from a double-strand structure to a single-strand structure to facilitate replication of each strand consistent with the semiconservative model of DNA replication. DNA polymerases have highly-conserved structure, which means that their overall catalytic subunits vary, on a whole, very little from species to species. Conserved structures usually indicate evolutionary advantages. DNA polymerase is considered to be a holoenzyme since it requires a Magnesium ion as a co-factor to function properly. In the absence of the Magnesium ion, it is referred to as an apoenzyme. Error correction is a property of some, but not all, DNA polymerases. This process corrects mistakes in newly-synthesized DNA. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA. The 3'->5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following base excision, the polymerase can re-insert the correct base and replication can continue. Some viruses also encode special DNA polymerases which may selectively replicate viral DNA through a variety of mechanisms. Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase (RdDp). It polymerizes DNA from a template of RNA. 4- DNA Modifying Enzymes Remove or add chemical groups. e.g.: T4 PNKase: 5-ends labelling of DNA and RNA molecules.

5- Topoisomerases Introduce or remove supercoils from covalently closed circular DNA.