DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy fghtheir DNA. This process is "replication" in that each strand of the original double-stranded DNA molecule serves as template for the reproducfgtion of the complemensdftary strand. Hence, following DNA replication, two identical DNA moleculxcves have been produced from a single double-stranded DNA molecule. Cellular proofreading and error toe-checking mechanisms ensure near perfect fidelity for DNA replication.
In a cell, DNA replication begins at specific locations in the genome, called "origins". Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis.
DNA replication can also be performed in vitro (outside a cell). DNA polymerases, isolated from cells, and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA..
DNA usually exists as 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: adenine, cytosine, guanine, and thymine. A nucleotide is a mono-, di- or triphosphate deoxyribonucleoside; that is, a deoxyribose sugar is attached to one, two or three phosphates . Chemical interaction of these nucleotides forms phosphodiester linkages, creating the phosphate-deoxribose backbone of the DNA double helix with the bases pointing inward. Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine and cytosine pairs with guanine.
DNA strands have a directionality, and the different ends of a single strand are called the "3' (three-prime) end" and the "5' (five-prime) end." These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. In addition to being complementary, the two strands of DNA are antiparallel: they are oriented in opposite directions. This directionality has consequences in DNA synthesis, because DNA polymerase can only synthesize DNA in one direction by adding nucleotides to the 3' end of a DNA strand.
The pairing of bases in DNA through hydrogen bonding means that the information contained within each strand is redundant. The nucleotides on a single strand can be used to reconstruct nucleotides on a newly synthesized partner strand.
DNA polymerases are a family of enzymes that carry out all forms of DNA replication. A DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. To begin synthesis of a new strand, a short fragment of DNA or RNA, called a primer, must be created and paired with the template strand before DNA polymerase can synthesize new DNA.
Once a primer pairs with DNA to be replicated, DNA polymerase synthesizes 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 two of the three total phosphates attached to each unincorporated base. (Free bases with their attached phosphate groups are called nucleoside triphosphates.) When a nucleotide is being added to a growing DNA strand, two of the phosphates are removed and the energy produced creates a phosphodiester (chemical) bond that attaches the remaining phosphate to the growing chain. The energetics of this process also help explain the directionality of synthesis - if DNA were synthesized in the 3' to 5' direction, the energy for the process would come from the 5' end of the growing strand rather than from free nucleotides.
DNA polymerases are generally extremely accurate, making less than one error for every 107 nucleotides added. Even so, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a strand in order to correct mismatched bases. If the 5' nucleotide needs to be removed during proofreading, the triphosphate end is lost. Hence, the energy source that usually provides energy to add a new nucleotide is also lost.
For a cell to divide, it must first replicate its DNA. This process is initiated at particular points within the DNA, known as "origins", which are targeted by proteins that separate the two strands and initiate DNA synthesis. Origins contain DNA sequences recognized by replication initiator proteins (eg. dnaA in E coli' and the Origin Recognition Complex in yeast). These initiator proteins recruit other proteins to separate the two strands and initiate replication forks.
Initiator proteins recruit other proteins to separate the DNA strands at the origin, forming a bubble. Origins tend to be "AT-rich" (rich in adenine and thymine bases) to assist this process, because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair)—strands rich in these nucleotides are generally easier to separate due the positive relationship between the number of hydrogen bonds and the difficulty of breaking these bonds. Once strands are separated, RNA primers are created on the template strands. More specifically, the leading strand receives one RNA primer per active origin of replication while the lagging strand receives several; these several fragments of RNA primers found on the lagging strand of DNA are called Okazaki fragments, named after their discoverer. DNA polymerase extends the leading strand in one continuous motion and the lagging strand in a discontinuous motion (due to the Okazaki fragments). RNase removes the RNA fragments used to initiate replication by DNA Polymerase, and another DNA Polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.
As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming 2 replication forks. In bacteria, which have a single origin of replication on their circular chromosome, this process eventually creates a "theta structure" (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.
When replicating, the original DNA splits in two, forming two "prongs" which resemble a fork (hence the name "replication fork"). DNA has a ladder-like structure; imagine a ladder broken in half vertically, along the steps. Each half of the ladder now requires a new half to match it. Because DNA polymerase can only synthesize a new DNA strand in a 5' to 3' manner, the process of replication goes differently for the two strands comprising the DNA double helix.
