The Enzyme That Links DNA Fragments Together
DNA Fragments is composed of linear, polymer-like segments called chromosomes. The DNA polymerase enzyme only adds nucleotides in one direction; leading strand DNA synthesis continues until the chromosome reaches the end and lagging strand synthesis stops. The unpaired ends of chromosomes become shorter as cells divide. These unpaired ends have a repetitive sequence, called a telomere, which does not code for a specific gene.
ATP-dependent DNA ligase
ATP-dependent DNA ligases link DNA fragments together. The four types of ligases have distinct functions and structures, with some of them having domains similar to each other. DNA ligases IIIa, IIIb, and IV share the same general structure, and both have conserved non-catalytic domains. They all have a PCNA binding motif, mitochondrial targeting sequence, and BRCA carboxy-terminal-related domains.
The ATP-dependent DNA ligase I enzymes, nuclear DNA ligase IIIa and -IIIb, have a carboxy-terminal BRCT domain and interact with the mammalian DNA repair factor XrccI. DNA ligase IV, which also has a zinc-finger motif, is essential for non-homologous end-joining. Both enzymes have extensive sequence similarity to poly(ADP-ribose) polymerase II.
The evolutionary relationships of DNA ligases are not yet clear. Archaeal enzymes have similar primary structures but differ in nucleotide specificity. Archaeal DNA ligases, however, cannot utilize NAD+. Archaeal DNA ligases can be found in many organisms, including humans and fungi. However, they do not have a universal role in evolution, making it difficult to conclude.
ATP-dependent DNA ligases bind to single-stranded breaks in the phosphodiester backbone of double-stranded DNA. The ligase cleaves ATP into AMP and pyrophosphate and then covalently bonds these two to the nick’s 5′ phosphate. The ligand then seals the nick.
The ATP-dependent DNA ligase is a key enzyme for repairing damaged DNA. The enzyme can bind to adenylated or non-adenylated DNA fragments by binding to their termini. The process releases AMP, which subsequently links the fragments together. These proteins have been identified and studied by several groups. Their discovery is a watershed event in the field of molecular biology.
The ATP-dependent DNA ligase encoded by the lig gene in E. coli bacterium is an example of DNA ligase. This enzyme can bind blunt ends of DNA and oligonucleotides, but it cannot join DNA and RNA efficiently. It also requires ATP as a cofactor, so it is often difficult to detect without ATP.
Restriction enzymes are proteins that cut DNA at specific sites. They help researchers assemble and recombine fragments of DNA. Names of restriction enzymes derive from the species, genus, or strain designations of the bacterial organisms that produce them. They were originally thought to evolve from a common ancestral protein that recognized specific DNA sequences through gene amplification and genetic recombination.
Restriction enzymes are highly effective at joining fragments of DNA. They use billions of copies of DNA to make each product. These billions of copies bump into the restriction enzymes randomly, resulting in multiple products. The resulting joined products have distinct patterns. Restriction enzymes are highly specific for the types of DNA fragments cut by restriction enzymes. They are therefore important in repairing DNA breaks.
Restrictions are most effective in preparing DNA fragments for subsequence molecular cloning. Restriction digestion allows fragments to be pieced together to be analyzed using gel electrophoresis. The results are evaluated by comparing the products’ molecule lengths to their control samples. A typical reaction consists of a DNA template, restriction enzyme of choice, and buffer. The enzymes are incubated for specific amounts of time to ensure optimal activity. When a restriction enzyme is activated, the reaction is terminated with heat.
The discovery of the restriction enzymes in 1965 was an important step in developing genetic engineering. The enzymes helped scientists create self-propagating cloning vectors in E. coli, the backbone of many of today’s genetic engineering efforts. They also helped scientists make the first complete cDNA and the 32-kb polyketide synthase gene cluster.
The discovery of restriction enzymes came about thanks to the Loening paper. The enzymes were discovered through gel electrophoresis and now number over three thousand, representing 250 specificities. Since then, agarose gels and polyacrylamide slab-gel electrophoresis have been developed. In 1972, the visualization method was enhanced by the use of ethidium bromide.
Replication forks are located at the ends of the double-stranded DNA molecule. The forks move along the DNA helix, and each fork replicates ten base pairs, which correspond to one complete turn on the axis of the parental double helix. The forks require enormous energy to carry out the process, so an alternative strategy is developed. DNA topoisomerases are responsible for creating a swivel in the DNA helix.
Replication forks are crucial to the process of DNA replication. Replication forks are Y-shaped structures that initiate DNA synthesis and elongation. Replication forks are at the ends of a structure called the replication bubble, and an electron microscope observation of replicating DNA molecules reveals regions of DNA duplex splitting. These regions synthesize new DNA, and then the duplex splits back to a single strand.
The replication fork is a region of unwound DNA where the DNA polymerase and helper proteins can begin copying the DNA. Helicases catalyze the unwinding of the double helix at replication forks, and helicases create templates for DNA replication. The topoisomerase helps relieve the strain on the two strands.
Replication forks are the active site of the replication process. At the fork, DNA polymerase and helicase molecules act together to link two DNA fragments. Single strand binding proteins help facilitate the opening of the DNA helix. DNA polymerase on the leading strand can operate continuously, while the lagging strand must restart at short intervals. DNA primase molecule synthesizes short RNA primers to start the process.
Replication forks are involved in the linkage of DNA fragments. During the replication process, two strands of the parental DNA duplex separate. Then, two new daughter strands are synthesized. The new strands are identical, and the unreplicated part of the chromosome becomes smaller—afterward, DNA synthesis proceeds. Replication forks are important for the replication of DNA, and they are essential to the growth of many types of organisms.
Base pairing rules
DNA has a complementary base-pairing structure, which describes how each of the two strands binds to the other. For example, adenine binds to thymine by forming two hydrogen bonds, while guanine binds to cytosine by forming three hydrogen bonds. Then the enzyme DNA polymerase synthesizes new strands of DNA according to complementary base-pairing rules.
DNA polymerase uses complementary base-pairing rules to copy the DNA strand. Each strand contains two nucleotides, adenine, and thymine. The nucleotides must pair up for DNA to replicate. In addition, bases must be in the right order, or the copy will fail. DNA polymerase knows the order of nucleotides, but it cannot read the sequence of the other strand.
James Watson and Francis Crick developed a double helix model of DNA secondary structure. This model depicts two strands of DNA in an anti-parallel orientation and complementary primary nucleotide structures. These strands are colored red and green. The complementary strands are coiled together to form a double helix structure. The structure is further stabilized by the hydrophobic and pi-stacking interactions between the nucleotides.
The enzymes used to replicate DNA need the proper nucleotides to start the new polynucleotide chain. DNA polymerase cannot begin the new polynucleotide chain if the nucleotide is not base-paired. The enzyme cannot base-pair with the wrong nucleotide, making it useless as a template.