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DNA ligase is a type of enzyme 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 may specifically repair double-strand breaks. 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.
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones. These enzymes catalyze the chemical reaction
DNA topoisomerases are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA. Topological issues in DNA arise due to the intertwined nature of its double-helical structure, which, for example, can lead to overwinding of the DNA duplex during DNA replication and transcription. If left unchanged, this torsion would eventually stop the DNA or RNA polymerases involved in these processes from continuing along the DNA helix. A second topological challenge results from the linking or tangling of DNA during replication. Left unresolved, links between replicated DNA will impede cell division. The DNA topoisomerases prevent and correct these types of topological problems. They do this by binding to DNA and cutting the sugar-phosphate backbone of either one or both of the DNA strands. This transient break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed. Since the overall chemical composition and connectivity of the DNA do not change, the DNA substrate and product are chemical isomers, differing only in their topology.
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.
DNA polymerase I is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase. It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli Pol I enzyme is composed of 928 amino acids, and is an example of a processive enzyme — it can sequentially catalyze multiple polymerisation steps without releasing the single-stranded template. The physiological function of Pol I is mainly to support repair of damaged DNA, but it also contributes to connecting Okazaki fragments by deleting RNA primers and replacing the ribonucleotides with DNA.
DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA replication. It was discovered by Thomas Kornberg and Malcolm Gefter in 1970. The complex has high processivity and, specifically referring to the replication of the E.coli genome, works in conjunction with four other DNA polymerases. Being the primary holoenzyme involved in replication activity, the DNA Pol III holoenzyme also has proofreading capabilities that corrects replication mistakes by means of exonuclease activity reading 3'→5' and synthesizing 5'→3'. DNA Pol III is a component of the replisome, which is located at the replication fork.
In molecular biology and biochemistry, processivity is an enzyme's ability to catalyze "consecutive reactions without releasing its substrate".
Exodeoxyribonuclease V is an enzyme of E. coli that initiates recombinational repair from potentially lethal double strand breaks in DNA which may result from ionizing radiation, replication errors, endonucleases, oxidative damage, and a host of other factors. The RecBCD enzyme is both a helicase that unwinds, or separates the strands of DNA, and a nuclease that makes single-stranded nicks in DNA. It catalyses exonucleolytic cleavage in either 5′- to 3′- or 3′- to 5′-direction to yield 5′-phosphooligonucleotides.
In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically, while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ from exonucleases, which cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as "exo-endonucleases", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity.
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.
Taq polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism Thermus aquaticus, from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to Taq or Taq pol. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.
DNA polymerase II is a prokaryotic DNA-dependent DNA polymerase encoded by the PolB gene.
Exonuclease III (ExoIII) is an enzyme that belongs to the exonuclease family. ExoIII catalyzes the stepwise removal of mononucleotides from 3´-hydroxyl termini of double-stranded DNA. A limited number of nucleotides are removed during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules.
Exodeoxyribonuclease I is an enzyme that catalyses the following chemical reaction:
Prokaryotic DNA Replication is the process by which a prokaryote duplicates its DNA into another copy that is passed on to daughter cells. Although it is often studied in the model organism E. coli, other bacteria show many similarities. Replication is bi-directional and originates at a single origin of replication (OriC). It consists of three steps: Initiation, elongation, and termination.
Deoxyribonuclease IV (phage-T4-induced) is catalyzes the degradation nucleotides in DsDNA by attacking the 5'-terminal end.
Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The in vitro protein synthesis environment is not constrained by a cell wall or homeostasis conditions necessary to maintain cell viability. Thus, CFPS enables direct access and control of the translation environment which is advantageous for a number of applications including co-translational solubilisation of membrane proteins, optimisation of protein production, incorporation of non-natural amino acids, selective and site-specific labelling. Due to the open nature of the system, different expression conditions such as pH, redox potentials, temperatures, and chaperones can be screened. Since there is no need to maintain cell viability, toxic proteins can be produced.
T7 DNA polymerase is an enzyme used during the DNA replication of the T7 bacteriophage. During this process, the DNA polymerase “reads” existing DNA strands and creates two new strands that match the existing ones. The T7 DNA polymerase requires a host factor, E. coli thioredoxin, in order to carry out its function. This helps stabilize the binding of the necessary protein to the primer-template to improve processivity by more than 100-fold, which is a feature unique to this enzyme. It is a member of the Family A DNA polymerases, which include E. coli DNA polymerase I and Taq DNA polymerase.
The enzyme exodeoxyribonuclease VII is a bacterial exonuclease enzyme. It is composed of two nonidentical subunits; one large subunit and 4 small ones. that catalyses exonucleolytic cleavage in either 5′- to 3′- or 3′- to 5′-direction to yield nucleoside 5′-phosphates. The large subunit also contains an N-terminal OB-fold domain that binds to nucleic acids.
Exodeoxyribonuclease III is an enzyme that catalyses the following reaction
References
- ↑ Lindahl T, Gally JA, Edelman GM (February 1969). "Deoxyribonuclease IV: a new exonuclease from mammalian tissues". Proceedings of the National Academy of Sciences of the United States of America. 62 (2): 597–603. doi: 10.1073/pnas.62.2.597 . PMC 277851 . PMID 5256235.
- ↑ Little JW (February 1967). "An exonuclease induced by bacteriophage lambda. II. Nature of the enzymatic reaction". The Journal of Biological Chemistry. 242 (4): 679–86. doi: 10.1016/S0021-9258(18)96258-7 . PMID 6017737.
- ↑ "Lambda Exonuclease" (PDF). Epicentre.
