3. (a) Briefly compare and contrast the different types of restriction explain w

ID: 262474 • Letter: 3

Question

3. (a) Briefly compare and contrast the different types of restriction explain why Type II is the most useful in molecular biology. (b) Suggest condition(s) that may lead to partial digestion of DNA endonucleases and (4 marks) (2 marks) (c) If a circular plasmid having 2 EcoRI sites is partially digested by EcoRI, how many fragments and their forms (circular/linear) would you expect to see? If the M13 bacteriophage vector has only 1 EcoRI site, how many fragment(s) would 4 marks) you see if the conditions are set for complete digestion? 4.(a) What is the function of DNA methyltransferases? Briefly discuss their use in 4 marks) nature. (b) Give one example and suggest one application for the following enzymes (i) DNA-dependent polymerase (ii) RNA-dependent polymerase (ii) Template independent polymerase (6 marks)

Explanation / Answer

(3) a. There are four different types of restriction endonucleases (REs) which have been classified on the basis of their site of recognition (sequence and cleavge position) cofactor requirement and subunit composition. These are:

Protein structure

Recognition site

Cleavage site

Restriction and Methylation

Cofactors/ reaction requirements

TYPE

Bifunctional with 3 subunits

Bipartite and asymmetric

Non-specific, cuts at > 1000bp away from recognition site

Mutually exclusive

Both ATP and S-adenosyl methionine (SAM)

TYPE II

Separate endonuclease and methylase

4-6 bp palindromic sequences

At the same or close by to the recognition site

Separate reactions

Require Magnesium ions

TYPE III

Binfunctional enzyme with 2 subunits

5-7 bp asymmetric sequences

24-26 bp downstream to the recognition site

Simultaneous

Require ATP (do not hydrolyse it), SAM stimulates reaction

Other REs include TYPE IV (which cleave only methylated or modified DNA) and TYPE V target non- palindromic sequences with the help of guide RNAs.

TYPE II restriction enzymes are most useful in molecular biology due to their specific nature of cleaing at the same or close by to the palindromic recognition sites. Type II restriction enzyme are also independent of their methylase. Since majority of known restriction enzymes are of type II, they find the most use as laboratory tools. They produce discrete bands during gel electrophoresis, and are useful for DNA analysis and gene cloning. The first to be discovered and utilized was EcoRI, which is staggered and its recognition sequence is 5?-GAATTC-3?.

b. Partial digestion using restriction enzymes is done to generate all possible digetion fragments and is utilised during cloning. Partial digestion utilizes a lower amount of the enzyme to generate a mixture of fragments that have stochastically cleaved at restriction sites. It mainly depends on the amount of restriction enzyme, the concentration of DNA and its purity and the incubation otime of reaction. Reaction conditions such as temperature( affects conformation and activity) and UV irradiation ( makes sites inaccesible) can be used to modify the reaction rate.

(c) In case of a circular plasmid with 2 EcoRI sites, in case of partial digestion, all the sites may not be cleave dby the enzyme. Eco RI generates 4 nucleotide long sticky ends with 5' AATT overhangs. If both the sites in plasmid molecules are cleaved then it will generate 2 linear fragments( complete digestion). However, in partial digestion, psome plasmid mlecules would not be cleaved at both the sites and therefore due to sticky ends produced by EcoR restriction ( at single site), the molecule would reform to achieve a circuclar DNA form. Finally, 2 linear fragments and a circular DNA molecule would be observed during partial digestion.

In case of M13 bacteriophage which has only 1 EcoRI restriction site and is also a single stranded circular molecule, complete digestion would result in formation of a linear molecule as it will cut the vector at a single site.

4. (a) DNA methyltransferases are enzymes which utilize SAM as a donor of methyl group and transfer this methyl group to DNA. Methylation of DNA plays a critical role in epigenetics as well as in protection of DNA against viral infection in bacteria by making the restriction sites ( in bacterial genome) inaccessible to restriction enzymes. In maintenance of epigenome, the methyltransferases act by methylating at specific sites ( CpG islands in heterochromatin, lysine methylation, etc.) which helps in maintaining chromatin structure and helps in regulation of gene expression.

(b) (i) E. coli DNA Polymerase I is a DNA dependent Polymerase and is used as a repair polymerase involved in excision repair with both 3'–5' and 5'–3' exonuclease activity and processing of Okazaki fragments generated during lagging strand synthesis in DNA replication.

(ii) RNA replicase is a RNA-dependent RNA polymerase (RdRP)that catalyzes the replication of RNA from an RNA template in RNA viruses. RNA dependent DNA polymerase called reverse transcriptase is encoded by retroviruses. Being an RNA-dependent DNA polymerase (RdDp), it synthesizes DNA from a template of RNA. The reverse transcriptase contains both DNA polymerase functionality and RNase H functionality, which degrades RNA base-paired to DNA.

(iii) Terminal deoxynucleotidyl transferase works as a template independent polymera as it does not require a template and is mainly used in homopolymer tailing and labelling at 3' OH ends. It can work on rotruding, recessed or blunt-ended double or single-stranded DNA molecules as
substrates.