A number of molecular biologists originally believed that the first DNA molecule is the reason for life on earth. That sentiment changed after many years of research and experimental work. Now, biologists are quite convinced that DNA synthesis and replication requires a multitude of proteins, meaning DNA and its replication mechanisms must have appeared later on in the early history of life.
In fact, many molecular biologists believe that DNA originated in a world of RNA and protein instead, during a critical period of evolution (in the RNA world) and the emergence of the last universal common ancestor (LUCA, or LUA).
LUCA, a population of organisms from which all organisms living on earth are descended from, appears to have been a small, single-celled organism, possibly with a ring-shaped coil of DNA floating freely within the cell. This common ancestor, and its DNA, create a link from our cells back through thousands of generations. It almost sounds like an incredibly long game of shoots and ladders.
Now, deoxyribonucleic acid, or DNA, replicates. Its replication mechanisms explain how life as we know it evolved from single-celled organisms to something more complex. The genetic information stored is transferred and modified in organisms in several ways, making decoding our DNA and comparing it with other organisms critical to our understanding of the evolution of life on Earth.
The process of copying or duplicating genomic DNA is known as DNA replication. And the polymerization of new strands is performed using DNA polymerases, along with several other enzymes that are involved in the process. DNA polymerases are multi-subunit enzymes that catalyze the addition of nucleotides onto existing strands of DNA molecules. Arthur Kornberg first discovered this polymerase in 1956 in lysates of Escherichia coli (E.coli) after identifying their ability to synthesize a new DNA chain using deoxynucleotide triphosphates (dNTPs) and DNA template strands.
These enzymes are present in both eukaryotic and prokaryotic cells. Each organism contains many types of DNA pols, having distinct polymerase activity in different biochemical processes. Moreover, the number of replicative DNA polymerases in an organism shows the complexity of its genome and replication process. For example, eukaryotes have larger genomes and more complex regulations than prokaryotes. Thus, they possess a higher number of replicative and repair DNA pols (including organelle-specific enzymes) than prokaryotes and viruses.
This article provides an overview of the DNA polymerases’ structures and functions in both eukaryotes and prokaryotes.
More complex cells possess more DNA polymerases; viruses have only 1 DNA pol, prokaryotes have 5 DNA pol (DNA polymerase I, II, III, IV, and V), and eukaryotes have 15 DNA pol, designated with greek letters as DNA pol α, 𝛃, 𝛄, ẟ, ε, ζ, η, θ, ι, 𝛋, λ. 𝛍, and v.
DNA polymerases from all living organisms have been isolated and studied. Based on their sequence similarity, they are classified into seven families: A, B, C, D, X, Y, and RT. The structural characterization of the four main families of DNA polymerases are given below:
A-family DNA polymerases consist of either replicative or repair polymerases. During the catalytic process of DNA synthesis, replicative polymerases match free nucleotides to template DNA strands. A base-pairing (guanine (G) base pairs with cytosine (C), and adenine (A) base pairs with thymine (T)) occurs between the two strands with the formation of hydrogen bonds. Its examples include T7 DNA polymerase and the eukaryotic mitochondrial DNA polymerase γ.
The repair polymerase checks or proofreads the DNA for mutagenesis or abnormalities in the DNA strand. They show both 5’-3’ and 3’-5’ (in some) exonuclease activity. The repair polymerases are DNA pol I from E. coli, pol I from Thermus aquaticus, and pol I from Bacillus stearothermophilus.
DNA pol I of E.coli has both 5′→3′ polymerase domains and 3′→5′ exonuclease domains. These features allow the polymerase to function in gaps filling, arise during DNA replication and recombination processes.
B-family DNA polymerases are involved in processing DNA replication. They catalyze the synthesis of both the leading strand and the lagging strand, which is synthesized as small oligonucleotides called Okazaki fragments of DNA.
The incorporation of any wrong nucleotides can lead to several mutations. To correct these mistakes, B-family DNA polymerases perform a proofreading activity that helps in the identification of any errors in the newly synthesized DNA. The family of these polymerases is present in fungi, plants, and some bacteriophages. Its example includes DNA pol II.
Family X consists of eukaryotes’ DNA polymerase pol β (main enzymatic activity in base excision repair), pol μ, pol λ, and pol σ. Pol β helps to repair alkylated, oxidized, or abasic sites in the DNA strands. Pol λ and pol μ are involved in repairing damaged sites due to hydrogen peroxide and ionizing radiation.
Y-family DNA polymerases contain pol IV, which is an error-prone DNA polymerase involved in non-targeted mutagenesis. Pol IV is expressed by the dinB gene after a DNA damage response. This signal interferes with pol III holoenzyme processivity, which stops replication and gives time for DNA repair.
The Y-family of DNA polymerases also contains pol V that bypass DNA damage to continue replication.
DNA polymerases perform two central functions in organisms: DNA replication and DNA repair. Both activities are essential to the proper development and function of an organism.
During in vivo DNA replication, the first step is to unwind a DNA double helix and disrupt the hydrogen bonds so that there are two single strands. This results in the formation of a Y-shaped structure called a replication fork. Each strand exposes the original site for initiation of replication.
DNA replication is a discontinuous process, thus requiring more than one DNA pol to work on leading and lagging strands. In eukaryotes, the process is initiated by primase (DNA-dependent RNA polymerase) that synthesizes a short oligonucleotide RNA sequence on the template DNA, called a primer.
The RNA sequence is then transferred to the active site of DNA pol α, which initiates replication of DNA at the origins of replication (leading and lagging). Thereafter, DNA pol ẟ (DNA pol ε also plays a part) arrives at the strand, releasing DNA pol α, and completing the process of DNA replication by elongating the majority of the strand.
The DNA polymerases catalyze the linking of the 3′ hydroxyl group of the end nucleotide to the 5′ phosphate of nucleotide to be added.
Adenosine triphosphate (ATP) is also required to initiate and sustain DNA synthesis inside cells. It provides the energy required by enzymes to perform the process of replication.
In lab conditions, PCR is used to make millions of copies of DNA utilizing a specific DNA sample. A mixture of reagents or substrates is used, including:
A high-fidelity PCR, which involves either a blend of Taq polymerase, proofreading enzymes, or high-fidelity polymerase, is used to prevent errors during in vitro replication.
The PCR assay has application in several research studies, including sequencing, gene cloning and manipulation, gene mutagenesis, diagnosis and monitoring of genetic disorders, and analysis of genetic fingerprints for DNA profiling.
Image: An illustration of a single PCR cycle.
The DNA repair polymerases are essential to maintain the integrity of genomes. They are involved in repairing single-strand and double-strand breaks, strand cross-linking, base loss, and base modifications.
Different polymerases have different biochemical properties that allow them to follow various repair pathways, which include mismatch repairs, nucleotide excision repairs, base excision repairs, double-strand break repairs, and interstrand cross-link
repairs.
Image: An illustration of nucleotide excision repair.
Excedr’s lease program has provided a creative financial solution for many laboratories working on a budget. Lease the cutting-edge lab equipment you need, save capital for other parts of your business, and achieve R&D milestones quicker.
DNA polymerases are one of the essential catalytic enzymes involved in DNA replication in different organisms. These enzymes have very different structural and functional properties in different organisms. They are topics of massive interest in many life science laboratories, including biochemistry, molecular biology, and biotechnology labs.
Performing experiments at this molecular level requires several advanced tools and machines.
We bring you budget-friendly custom lease programs. At Excedr, you can lease the cutting-edge lab equipment and resources needed for your R&D without the expensive upfront costs.
Contact one of our leasing reps if you have any queries. We’ll be able to help you right away!
