DNA replication
DNA replication is the process by which a cell makes exact copies of its DNA. This process occurs in all organisms and is essential to biological inheritance, cell division, and repair of damaged tissues. DNA replication ensures that each of the newly divided daughter cells receives its own copy of each DNA molecule.
DNA most commonly occurs in double-stranded form, made up of two complementary strands held together by base pairing of the nucleotides comprising each strand. The two linear strands of a double-stranded DNA molecule typically twist together in the shape of a double helix. During replication, the two strands are separated, and each strand of the original DNA molecule then serves as a template for the production of a complementary counterpart strand, a process referred to as semiconservative replication. As a result, each replicated DNA molecule is composed of one original DNA strand as well as one newly synthesized strand. Cellular proofreading and error-checking mechanisms ensure near-perfect fidelity for DNA replication.
DNA replication usually begins at specific locations known as origins of replication which are scattered across the genome. Unwinding of DNA at the origin is accommodated by enzymes known as helicases and results in replication forks growing bi-directionally from the origin. Numerous proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by incorporating nucleotides that complement the nucleotides of the template strand. DNA replication occurs during the S stage of interphase.
DNA replication can also be performed in vitro. DNA polymerases isolated from cells and artificial DNA primers can be used to start DNA synthesis at known sequences in a template DNA molecule. Polymerase chain reaction, ligase chain reaction, and transcription-mediated amplification are all common examples of this technique. In March 2021, researchers reported evidence suggesting that a preliminary form of transfer RNA, a necessary component of translation, could have been a replicator molecule itself in the early abiogenesis of primordial life.
DNA structure
DNA is a double-stranded structure, with both strands coiled together to form the characteristic double helix. Each single strand of DNA is a chain of four types of nucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four nucleobases: adenine, cytosine, guanine, and thymine, commonly abbreviated as A, C, G, and T, respectively. Adenine and guanine are purine nucleobases, while cytosine and thymine are pyrimidines. These nucleotides form phosphodiester bonds, creating phosphate-deoxyribose backbone of the DNA double helix with the nucleobases pointing inward. Complementary nucleobases are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine, and guanine pairs with cytosine.DNA strands have a directionality, and the different ends of a single strand are called the "3′ end" and the "5′ end". By convention, if the base sequence of a single strand of DNA is given, the left end of the sequence is the 5′ end, while the right end of the sequence is the 3′ end. The strands of the double helix are anti-parallel, with one being 5′ to 3′, and the opposite strand 3′ to 5′. These terms refer to the chemical convention of numbering the carbon atoms comprising the deoxyribose molecule and indicate the specific carbon atom to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3′ end of a DNA strand.
The pairing of complementary bases in DNA means that the information contained within each strand is redundant. Phosphodiester bonds are stronger than hydrogen bonds. The actual job of the phosphodiester bonds is to connect the 5' carbon atom of one nucleotide to the 3' carbon atom of another nucleotide, while the hydrogen bonds stabilize DNA double helices across the helix axis but not in the direction of the longitudinal axis. This makes it possible to separate the strands from one another. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand.
DNA polymerase
s are a family of enzymes that carry out all forms of DNA replication. DNA polymerases in general cannot initiate synthesis of new strands but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand.DNA polymerase adds a new strand of DNA by extending the 3′ end of an existing nucleotide chain, adding new nucleotides matched to the template strand, one at a time, via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from hydrolysis of the high-energy phosphate bonds between the three phosphates attached to each unincorporated base. Free bases with an attached sugar molecule are called nucleosides, and nucleosides with one or more attached phosphate groups are called nucleotides; in particular, nucleosides with three attached phosphate groups are called nucleoside triphosphates. When a free nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the free nucleotide and the deoxyribose of another nucleotide within the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the free nucleotide's two distal phosphate groups as a pyrophosphate. Enzymatic hydrolysis of the resulting pyrophosphate into inorganic phosphate consumes a second high-energy phosphate bond and renders the reaction effectively irreversible.
In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added. Some DNA polymerases can also delete nucleotides from the end of a developing strand in order to fix mismatched bases. This is known as proofreading. Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 109 nucleotides added.
The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli. During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.
Replication process
DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination.Initiation
For a cell to divide, it must first replicate its DNA. DNA replication is an all-or-none process; once replication begins, it proceeds to completion. Once replication is complete, it does not occur again in the same cell cycle. This is made possible by the division of initiation of the pre-replication complex.Pre-replication complex
In late mitosis and early G1 phase, a large complex of initiator proteins assembles into the pre-replication complex at particular points in the DNA, known as "origins". In E. coli the primary initiator protein is Dna A; in yeast, this is the origin recognition complex. Sequences used by initiator proteins tend to be "AT-rich", because A-T base pairs have two hydrogen bonds and thus are easier to strand-separate. In eukaryotes, the origin recognition complex catalyzes the assembly of initiator proteins into the pre-replication complex. In addition, a recent report suggests that budding yeast ORC dimerizes in a cell cycle dependent manner to control licensing. In turn, the process of ORC dimerization is mediated by a cell cycle-dependent Noc3p dimerization cycle in vivo, and this role of Noc3p is separable from its role in ribosome biogenesis. and Noc3p are continuously bound to the chromatin throughout the cell cycle. Cdc6 and Cdt1 then associate with the bound origin recognition complex at the origin in order to form a larger complex necessary to load the Mcm complex onto the DNA. In eukaryotes, the Mcm complex is the helicase that will split the DNA helix at the replication forks and origins. The Mcm complex is recruited at late G1 phase and loaded by the ORC-Cdc6-Cdt1 complex onto the DNA via ATP-dependent protein remodeling. The loading of the MCM complex onto the origin DNA marks the completion of pre-replication complex formation.If environmental conditions are right in late G1 phase, the G1 and G1/S cyclin-Cdk complexes are activated, which stimulate expression of genes that encode components of the DNA synthetic machinery. G1/S-Cdk activation also promotes the expression and activation of S-Cdk complexes, which may play a role in activating replication origins depending on species and cell type. Control of these Cdks vary depending on cell type and stage of development. This regulation is best understood in budding yeast, where the S cyclins Clb5 and Clb6 are primarily responsible for DNA replication. Clb5,6-Cdk1 complexes directly trigger the activation of replication origins and are therefore required throughout S phase to directly activate each origin.
In a similar manner, Cdc7 is also required through S phase to activate replication origins. Cdc7 is not active throughout the cell cycle, and its activation is strictly timed to avoid premature initiation of DNA replication. In late G1, Cdc7 activity rises abruptly as a result of association with the regulatory subunit DBF4, which binds Cdc7 directly and promotes its protein kinase activity. Cdc7 has been found to be a rate-limiting regulator of origin activity. Together, the G1/S-Cdks and/or S-Cdks and Cdc7 collaborate to directly activate the replication origins, leading to initiation of DNA synthesis.