03/09/2013 15:08

1) DNA Ligase

In molecular biology, DNA Ligase is a specific type of enzyme, a ligase,  that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.

It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such as DNA ligase IV) may specifically repair double-strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA.

The first DNA ligase was purified and characterized in 1967. The common commercially available DNA ligases were originally discovered inbacteriophage T4, E. coli and other bacteria.

Ligase mechanism

The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). ATP is required for the ligase reaction, which proceeds in three steps:

1.    adenylation (addition of AMP) of a lysine residue in the active center of the enzyme, pyrophosphate is released;

2.    transfer of the AMP to the 5' phosphate of the so-called donor, formation of a pyrophosphate bond;

3.    formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor.    

Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required.

E. coli DNA ligase

The E. coli DNA ligase is encoded by the lig gene. DNA ligase in E. coli, as well as most prokaryotes, uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. It does not ligate blunt-ended DNA except under conditions of molecular crowding with polyethylene glycol, and cannot join RNA to DNA efficiently.

T4 DNA ligase

The DNA ligase from bacteriophage T4 is the ligase most-commonly used in laboratory. It can ligate cohesive ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate blunt-ended DNA with much greater efficiency than E. coli DNA ligase. Unlike E. coli DNA ligase, T4 DNA ligase cannot utilize NAD and it has an absolute requirement for ATP as a cofactor.

Applications in molecular biology research

Controlling the optimal temperature is a vital aspect of performing efficient recombination experiments involving the ligation of cohesive-ended fragments. Most experiments use T4 DNA Ligase (isolated from bacteriophag T4), which is most active at 37°C. However, for optimal ligation efficiency with cohesive-ended fragments ("sticky ends"), the optimal enzyme temperature needs to be balanced with the melting temperature T_m of the sticky ends being ligated, the homologous pairing of the sticky ends will not be stable because the high temperature disrupts hydrogen bonding. A ligation reaction is most efficient when the sticky ends are already stably annealed, and disruption of the annealing ends would therefore result in low ligation efficiency. The shorter the overhang , the lower the T_m.

Since blunt-ended DNA fragments have no cohesive ends to anneal, the melting temperature is not a factor to consider within the normal temperature range of the ligation reaction. However, the higher the temperature, the lower the chance that the ends to be joined will be aligned to allow for ligation (molecules move around the solution more at higher temperatures). The limiting factor in blunt end ligation is not the activity of the ligase but rather the number of alignments between DNA fragment ends that occur. The most efficient ligation temperature for blunt-ended DNA would therefore be the temperature at which the greatest number of alignments can occur. The majority of blunt-ended ligations are carried out at 14-25°C overnight. The absence of stably annealed ends also means that the ligation efficiency is lowered, requiring a higher ligase concentration to be used.

Alkaline phosphatase (ALP, ALKP)  is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation.

As the name suggests, alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphatase.

Typical use in the lab for alkaline phosphatases includes removing phosphate monoester to prevent self ligation.

Common alkaline phosphatases used in research include:

·         Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis). This phosphatase is easily inactivated by heat, a useful             feature in some applications.

·         Calf-intestinal alkaline phosphatase (CIP)

·         Placental alkaline phosphatase (PALP) and its C terminally truncated version that lacks the last 24 amino acids (constituting the domain that targets for GPI membrane anchoring) - the secreted alkaline phosphatase (SEAP)

Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5' end.

Removing these phosphates prevents the DNA from ligating (the 5' end attaching to the 3' end), thereby keeping DNA molecules linear until the next step of the process for which they are being prepared; also, removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment. For these purposes, the alkaline phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.

3) S1 Nuclease (Aspergillus nuclease S1)

Aspergillus nuclease S1 is an endonuclease enzyme derived from Aspergillus oryzae that splits single-stranded DNA (ssDNA) and RNA into oligo- or mononucleotides. 

Although its primary substrate is single-stranded, it can also occasionally introduce single-stranded breaks in double-stranded DNA or RNA, or DNA-RNA hybrids.

The enzyme hydrolyses single stranded region in duplex DNA such as loops or gaps.

Nomenclature

Alternative names include, endonuclease S1 (Aspergillus), single-stranded-nucleate endonuclease, deoxyribonuclease S1, deoxyribonuclease S1, nuclease S1, Neurospora crassa single-strand specific endonuclease, S1 nuclease, single-strand endodeoxyribonuclease, single-stranded DNA specific endonuclease, single-strand-specific endodeoxyribonuclease, single strand-specific DNase and Aspergillus oryzae S1 nuclease.

Properties

Aspergillus nuclease S1 is a monomeric protein of a molecular weight of 38 kilodalton.

It requires Zn²⁺ as a cofactor and is relatively stable against denaturing agents like urea, SDS, or formaldehyde.

The optimum pH for its activity lies between 4-4.5.

Aspergillus nuclease S1 is known to be inhibited somewhat by 50 μM ATP and nearly completely by 1 mM ATP. 50% inhibition has been shown at 85 μM dAMP and 1 μM dATP but uninhibited by cAMP.

Uses

Aspergillus nuclease S1 is used in the laboratory as a reagent in nuclease protection assays.

In molecular biology, it is used in removing single stranded tails from DNA molecules to create blunt ended molecules and opening hairpin loops generated during synthesis of double stranded cDNA.

4) Terminyl Transferase (Terminal Deoxynucleotidyltransferase)

Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells.

Function

TdT catalyses the addition of nucleotides to the 3' terminus of a DNA molecule. Unlike most DNA polymerases it does not require a template and is thus an example of a DNA polymerase that does not require a primer. The preferred substrate of this enzyme is a 3'-overhang, but it can also add nucleotides to blunt or recessed 3' ends. Cobalt is a necessary cofactor, however the enzyme catalyzes reaction upon Mg and Mn administration in vitro.It can also add homopolymers of ribonucleotides to the 3' end of DNA.

Terminal transferase is useful for at least two procedures:

Labeling the 3' ends of DNA: Most commonly, the substrate for this reaction is a fragment of DNA generated by digestion with a restriction enzyme that leaves a 3' overhang, but oligodeoxynucleotides can also be used. When such DNA is incubated with tagged nucleotides and terminal transferase, a string of the tagged nucleotides will be added to the 3' overhang or to the 3' end of the oligonucleotide.  

Adding complementary homopolymeric tails to DNA: This clever procedure was commonly used in the past to clone cDNAs into plasmid vectors, but has largely been replaced by other, much more efficient techniques. The principles of this technique are depicted in the figure below. Basically, terminal transferase is used to tail a linearized plasmid vector with G's and the cDNA with C's. When incubated together, the compementary G's and C's anneal to "insert" the cDNA into the vector, which is then transformed into E. coli.

Terminal transferase has applications in molecular biology.

It can be used in RACE to add nucleotides which can then be used as a template for a primer in subsequent PCR.

It can also be used to add nucleotides labeled with radioactive isotopes, for example in the TUNEL assay(Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) for the demonstration of apoptosis (which is marked, in part, by fragmented DNA).

5) Reverse Transcriptase

Reverse transcriptase is a common name for an enzyme that functions as a RNA-dependent DNA polymerase. T

hey are encoded by retroviruses, where they copy the viral RNA genome into DNA prior to its integration into host cells.

Reverse transcriptases have two activities:

• DNA polymerase activity: In the retroviral life cycle, reverse transcriptase copies only RNA, but, as used in the laboratory, it will transcribe both single-stranded RNA and single-stranded DNA templates with essentially equivalent efficiency. In both cases, an RNA or DNA primer is required to initiate synthesis.
• RNase H activity: RNase H is a ribonuclease that degrades the RNA from RNA-DNA hybrids, such as are formed during reverse transcription of an RNA template. This enzyme functions as both an endonuclease and exonuclease in hydrolyzing its target.

All retroviruses have a reverse transcriptase, but the enzymes that are available commercially are derived from one of two retroviruses, either by purification from the virus or expression in E. coli:

• Moloney murine leukemia virus: a single polypeptide
• Avian myeloblastosis virus: composed of two peptide chains

Applications in Molecular Biology Research

Reverse transcriptase is used to copy RNA into DNA. This task is integral to cloning complementary DNAs (cDNAs), which are DNA copies of mature messenger RNAs. Cloning cDNAs-the technique is usually initiated by mixing short (12-18 base) polymers of thymidine (oligo dT) with messenger RNA such that they anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo dT as a primer to synthesize so-called first-strand cDNA.

 

Another common use for reverse transcriptase is to generate DNA copies of RNAs prior to amplifying that DNA by polymerase chain reaction (PCR). Reverse Transcription PCR, usually called simply RTPCR, is a stupifyingly useful tool for such things as cloning cDNAs, diagnosing microbial diseases rapidly and a myriad of other applications. In most cases, standard preparations of reverse transcriptase are used for RTPCR, but mutated forms with relatively high thermal stability have been developed to facilitate the process.

6) DNA Polymerases

A DNA polymerase is a cellular or viral polymerase enzyme that synthesizes DNA molecules from their nucleotide building blocks.

DNA polymerases are essential for DNA replication, and usually function inpairs while copying one double-stranded DNA molecule into two double-stranded DNAs in a process termed semiconservative DNA replication.

DNA polymerases also play key roles in other processes within cells, including DNA repair, genetic recombination, reverse transcription, and the generation of antibody diversity via the specialized DNA polymerase, terminal deoxynucleotidyl transferase. 

In 1956, Arthur Kornberg and colleagues discovered the enzyme DNA polymerase I, also known as Pol I, in Escherichia coli. DNA polymerase II was also discovered by Kornberg and Malcolm E. Gefter in 1970 while further elucidating the role of Pol I in E. coli DNA replication.

Function

DNA polymerase can add free nucleotides only to the 3' end of the template strand. This results in elongation of the newly forming strand in a 5'-3' direction. No known DNA polymerase is able to begin a new chain (de novo).

DNA polymerase can add a nucleotide only on to a pre-existing 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Primers consist of RNA and/or DNA bases. In DNA replication, the first two bases are always 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.

It is important to note that the directionality of the newly forming strand (the daughter strand) is opposite to the direction in which DNA polymerase moves along the template strand. Since DNA polymerase requires a free 3' OH group for initiation of synthesis, it can synthesize in only one direction by extending the 3' end of the preexisting nucleotide chain. Hence, DNA polymerase moves along the template strand in a 3'-5' direction, and the daughter strand is formed in a 5'-3' direction. This difference enables the resultant double-stranded DNA formed to be composed of two DNA strands which are antiparallel to each other.

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 moves backwards 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 forwards.

 

Various DNA polymerases are extensively used in molecular biology experiments.

DNA polymerases are widely used in molecular biology laboratories, notably for the polymerase chain reaction (PCR), DNA sequencing, and molecular cloning.

7) Ribonuclease H

The enzyme RNase H is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism.

Members of the RNase H family can be found in nearly all organisms, from archaea to bacteria and eukaryota.

RNase H’s ribonuclease activity cleaves the 3’-O-P bond of RNA in a DNA/RNA duplex to produce 3’-hydroxyl and 5‘-phosphate terminated products.

In DNA replication, RNase H is responsible for removing the RNA primer, allowing completion of the newly synthesized DNA.

Function

In a molecular biology laboratory, as RNase H specifically degrades the RNA in RNA:DNA hybrids and will not degrade DNA or unhybridized RNA, it is commonly used to destroy the RNA template after first-strandcomplementary DNA (cDNA) synthesis by reverse transcription, as well as procedures such as nuclease protection assays.

RNase H can also be used to degrade specific RNA strands when the cDNA oligo is hybridized, such as the removal of the poly(A) tail from mRNA hybridized to oligo(dT), or the destruction of a chosen non-coding RNA inside or outside the living cell. To terminate the reaction, a chelator, such as EDTA, is often added to sequester the required metal ions in the reaction mixture

Sources:

Content:

https://en.wikipedia.org/wiki/DNA_ligase

https://en.wikipedia.org/wiki/Alkaline_phosphatase

https://en.wikipedia.org/wiki/Aspergillus_nuclease_S1

https://en.wikipedia.org/wiki/Terminal_deoxynucleotidyl_transferase

https://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/tt.html

https://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/rt.html

https://en.wikipedia.org/wiki/DNA_polymerase

 

https://en.wikipedia.org/wiki/RNase_H

Images:

https://fhs-bio-wiki.pbworks.com/w/page/12145760/DNA%20structure

https://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/tt.html

https://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/rt.html

https://www.neb.com/tools-and-resources/feature-articles/anatomy-of-a-polymerase-how-structure-effects-function