Uncovering Life's Building Blocks: Exploring Molecular Biology - Essay Sample

Introduction

Molecular biology is a biological discipline whose objective is to study the functions and structure of such life’s essential macromolecules like nucleic acids and proteins. In that case, therefore, it seeks to understand life right from its tiniest building blocks (Pecorino, 2012). By so doing, it creates an avenue through which scientist understand complex life processes. For instance, it is out of molecular biology that experts have been able to come up with the literature on such aspects as DNA (Kamps-Hughes, Quimby, Zhu, and Johnson, 2013). Moreover, the discipline mentioned above has also enabled researchers to gather enough information around the mode of action surrounding Type II Restriction Endonucleases as we are going to see.

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Structure

We must understand the structure of the endonucleases mentioned above before diving into the description of the mode of action it portrays. Also, we need to take note of the fact that they exist in the form of either homodimeric or tetrameric enzymes. Imperatively, therefore, type II restriction endonucleases refer to components of restriction-modification (RM) systems. That means that it entails a methyltransferase and an endonuclease activity (Pingoud, 2012). It is also critical to take note of the fact that they are the components that facilitate the systems that inhibit archaea and bacteria from attacking foreign DNA.

Mode of Action

Typically, type II restriction endonucleases cleave DNA at specific positions as per their recognition sequence. As a result, it creates different gel electrophoresis patterns and reproducible fragments (Pingoud, Wilson, and Wende, 2016). Then the endonucleases mentioned above proceeds to form homodimers containing recognition points. Notably, the resulting homodimers are palindromic and undivided with 4-8 long nucleotides. After that, they identify and cleave DNA at similar sites without the use of AdoMet or ATP since they generally use Mg2+ as the cofactor (Pingoud et al., 2016). In the process, the endonucleases also cleave the phosphodiester bonds of a double helix DNA. At times it may also cleave at a staggering site leaving behind overhangs known as sticky ends. Alternatively, it cleaves in the middle of both strands, thereby yielding blunt ends. Also, Type IIB restriction endonucleases (such as BpII and Bcgl) are generally multimers, bearing over one subunit (Pingoud et al., 2016). They cut out the site of recognition through cleaving DNA on both ends of identification. Notably, the endonucleases require both Mg2+ and AdoMet cofactors.

On the other hand, type IIE restriction endonucleases (such as Nael) tend to cleave the DNA after interacting with two sets of their recognition sites. One recognition sequence functions as the cleavage target whereas the other plays the role of an allosteric effector thereby speeding up or enhancing the effectiveness of enzyme cleavage (Lubys, Vitkute, Lubiene, and Janulaitis, 2013). Technically, type IIF restriction endonucleases (such as NgoMIV) function like type IIE endonucleases. That means that they interact with two sets of their recognition sequences but cleave the sequences at simultaneous.

Notably, type IIG restriction endonucleases lack such subunits as Eco57I. However, Type II restriction endonucleases but need cofactor AdoMet to be active (Lubys et al., 2013). There is also type IIM restriction endonucleases, (e.g., DpnI) which is capable of identifying and cutting methylated DNA. Additionally, type IIS restriction endonucleases such as FokI work by cleaving DNA at defined distances from their non-palindromic asymmetric recognition points; the characteristic is mostly applied in performing in-vitro cloning techniques and Golden Gate cloning. The enzymes could act as dimers (Lubys, Vitkute, Lubiene, and Janulaitis, 2013). Likewise, Type IIT restriction endonucleases (such as BslI and Bpu10I) are made up of two distinct subunits. Some of the units identify palindromic sequences, whereas others are composed of asymmetric recognition sites.

Applications of Type II Restriction Endonucleases

Gene cloning is one area that heavily relies on type II restriction endonucleases. Typically, it entails the in vitro production of novel DNA molecules which consist of new combinations of oligonucleotides or genes as well as the propagation of recombinant DNA molecules by exploiting in vitro replicative bacterial mechanisms and other microorganisms (Recombinant DNA and Gene Cloning - Biology LibreTexts, 2020). The emergence of genetic engineering methods has enabled the modification of microbial genome to produce elements of minute intrinsic importance but of immense medical value to human beings. The fact that DNA cloning involves making similar copies of specific pieces of DNA highlights the need for type II restriction endonucleases in the process. The DNA or gene is first put into a circular molecule known as a plasmid then the mechanism of type II restriction endonucleases is responsible for insertion through recognizing, cutting, and pasting the DNA, thereby producing a particle of recombinant DNA (Recombinant DNA and Gene Cloning - Biology LibreTexts, 2020). The recombinant plasmid is then introduced into a bacterium. The bacteria transporting the plasmid is then identified and grown. During the reproduction, the plasmid is replicated and passed to the offspring, generating sets of the DNA contained in them. It is worth noting that DNA cloning has resulted in lots of knowledge through permitting research and sequencing of particular genes from various organisms. Foreign genes are being inserted into the E. coli DNA with the view of enabling the synthesis of essential proteins. Human hormones like somatotropin, insulin, and somatostatin have been produced in E. coli.

Golden Gate cloning is a technique that applies the use of type II restriction endonucleases. It permits researchers to directionally and instantaneously gather various DNA fragments into one molecule. It does so by using T4 DNA ligase and Type IIs restriction endonucleases (Engler, and Marillonnet, 2014). The assembly is conducted in vitro. Mostly, the technique utilizes Type IIS endonucleases used are; BbsI, BsaI, and BsmBI. Biologically, its mechanism contrasts those of other types II restriction endonucleases since its enzymes cut the DNA outside their recognition points. Hence it can result in non-palindromic overhangs. There are 256 probable overhang sequences possible. Thus, various fragments of DNA may be gathered by the use of overhang sequence combinations (Engler, and Marillonnet, 2014). Golden Gate cloning is hence scarless.

Furthermore, because the end product lacks a Type II restriction endonuclease recognition site, a correctly-ligated element may not be cut after that by the restriction endonuclease hence the reaction is irreversible (Engler, and Marillonnet, 2014). Whereas the Golden Gate cloning technique may be employed in single insertion, scholars have used the method in assembling various DNA pieces simultaneously. The approach enables the construction of multigene formations for plant transformations.

Type II restriction endonucleases are essential in accomplishing proper DNA fragmentation and analysis. In an era where only a minute quantity of sperms can result in pregnancy through intracytoplasmic sperm injection or in vitro fertilization, identifying healthy sperms is important (Robinson et al., 2012). Sperm DNA fragmentation (SDF) has been identified to be higher amongst infertile men. Consequently, alkaline comet tests and Terminal deoxynucleotidyl deoxyuridine, and triphosphate nick-end labelling (TUNEL) are notable SDF analysis techniques that quantify DNA damage. They also correlate with assisted reproduction tests as compared to indirect apparatus like sperm chromatin structure assay (Robinson et al., 2012). Type II restriction endonucleases come in handy when separating DNA strands into pieces with the aim of selecting the fertile particles, in what is referred to as the sperm DNA fragmentation test. The test offers a dependable examination of sperm DNA integrity thereby helping in the identification of men who are likely to fail in initiating a healthy pregnancy (Carvalho, Norouzy, Ribeiro, Nau, & Pischel, 2015). Sperm DNA integrity helps in clinical diagnosis and treatment of infertility among men and could be of analytical value when assessing the outcome of assisted conception treatments. Identifying high quantities of DNA fragmentation in sperms may guide a clinician to determine whether an individual qualifies for sperm donation. Sperm DNA fragmentation tests also help couples in making informed decisions regarding their course of medication.

References

Bio.libretexts.org. 2020. 11.1: Recombinant DNA And Gene Cloning - Biology Libretexts. Available at: https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_Biology_(Kimball)/Unit_11%3A_Genomics/11.01%3A_Recombinant_DNA_and_Gene_Cloning

Carvalho, C. P., Norouzy, A., Ribeiro, V., Nau, W. M., & Pischel, U. (2015). Cucurbiturils as supramolecular inhibitors of DNA restriction by type II endonucleases. Organic & biomolecular chemistry, 13(10), 2866-2869.

Engler, C. and Marillonnet, S., 2014. Golden gate cloning. In DNA cloning and assembly methods (pp. 119-131). Humana Press, Totowa, NJ.

Kamps-Hughes, N., Quimby, A., Zhu, Z. and Johnson, E.A., 2013. Massively parallel characterization of restriction endonucleases. Nucleic acids research, 41(11), pp.e119-e119.

Lubys, A., Vitkute, J., Lubiene, J. and Janulaitis, A., Fermentas UAB, 2013. Restriction endonucleases and their applications. U.S. Patent 8,507,239.

Pecorino, L., 2012. Molecular biology of cancer: mechanisms, targets, and therapeutics. Oxford university press.

Pingoud, A. ed., 2012. Restriction endonucleases (Vol. 14). Springer Science & Business Media.

Pingoud, A., Wilson, G.G. and Wende, W., 2016. Type II restriction endonucleases—a historical perspective and more. Nucleic acids research, 44(16), p.8011.

Robinson, L., Gallos, I.D., Conner, S.J., Rajkhowa, M., Miller, D., Lewis, S., Kirkman-Brown, J. and Coomarasamy, A., 2012. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Human reproduction, 27(10), pp.2908-2917.