Introduction

The existence of cell division implies that there is a mechanism that replicates DNA and supplies identical copies for the daughter cells while still maintaining an accurate representation of the genome. This mechanism, known as DNA replication, occurs in all organisms and allows for genetic inheritance. It can occur in a short period, copying up to approximately ten to the 11th power (10^11) units of information in some cases. The process of replication is semi-conservative, meaning that each of the two DNA molecules formed from the process is made up of one, old, template strand and one newly formed strand. It also forms the basis of the expression of genetic information through protein synthesis. Considering DNA replication occurs rapidly, there are also various mechanisms to ensure correct replication and minimize errors. Cancers can arise from errors or mutations in DNA replication.[1][2][3]

Molecular Level

The blueprints of life are coded within the nucleic acid DNA (and for some organisms RNA). The structure of DNA is complex is made up of nucleotides, consisting of the sugar deoxyribose, a nitrogenous base (either adenine, guanine, cytosine, or thymine), and a phosphate group. Nucleotides made up of purine nitrogenous bases (adenine and guanine) can only pair with nucleotides made up of pyrimidine bases (thymine and cytosine, respectively). This isomorphism allows for base pairs to be replaced by one another and not ruin the backbone of the DNA. The monomers of the opposing strands stay together due to hydrogen bonds, and the two strands together form a double helix. Each strand runs antiparallel, meaning in opposite directions, one from the 5’ => 3’, the other 3’ => 5’ (This numbering comes from the carbon atoms in the sugar, which are labeled 1’ => 5’; the phosphate and hydroxyl group are attached to the 5’ and 3’ carbons respectively, creating the directionality of the nucleotide and, therefore, the DNA strand). The molecule is considered to be flexible, and that can be partially explained by the poor directionality of the hydrogen bonds which allow for bending or stretching. This flexibility enables many different DNA conformations, with high and low degrees of bending.

DNA is held in the nucleus of eukaryotes and within the cell membrane in the nucleoid for prokaryotes. It wraps around histone proteins to create nucleosomes, which group together to form chromatin and condense to form chromosomes. Chromatin can be negatively supercoiled (underwinded, more straightened) or positively supercoiled (overwinded, less straightened); this has implications in the use of DNA, either facilitating the binding of proteins to the nucleic acid or hindering it. The looser the DNA, the easier it is for proteins to access it, and the easier it is for replication to occur. Prokaryotic chromosomes are circular and are usually smaller in number. Eukaryotic chromosomes are linear and usually larger in number. Enzymes that are critical in DNA replication include DNA polymerase, helicase, topoisomerase, nuclease, ligase, and telomerase. These will be further elaborated on in the following sections.

Function

The function of DNA replication is multifold and essential to life as we know it. This biological process allows for the genetic blueprints of a cell to be passed on to daughter cells in cell division without loss of genetic information. Without replication, when the cell divides, the information would be split and only partially passed on.[4][5][6]

DNA replication also allows for protein synthesis, which is how genes are expressed. Protein synthesis begins with transcribing the specific gene, or section of DNA, which codes for the desired protein. Without replication, a gene could have a limited number of outputs and could transcribe a limited number of proteins.

Mechanism

Mechanism

The actual mechanism is brought by the interaction of a multitude of proteins and enzymes and occurs during the S phase. The single DNA strand is separated into two complementary strands. DNA replication is a semiconservative process, meaning that for every new pair there is one original strand and one new strand.

Initiation

The origin of replication is a sequence of base pairs in the genome where DNA replication begins; these sequences tend to be high in AT content making for easier separation. AT bonds have the fewest hydrogen bonds, making them weaker. Eukaryotes have multiple origins of replication whereas prokaryotes have one. Replication begins when origin-binding proteins bind to the origin of replication on the DNA. Then the unwinding of the double helix proceeds by way of the enzyme, helicase, creating a replication fork. The replication fork is Y-shaped and is where the leading and lagging strands are formed. Helicase begins unwinding by breaking hydrogen bonds. Single-stranded binding proteins (SSBs) stabilize the unwound DNA, preventing it from forming into secondary structures; the secondary structures can prevent the continuation of the DNA polymerase.

Elongation

Meanwhile, topoisomerases reduce the pressure on the winded portions and continue to open the DNA downstream to allow for elongation. They work by changing the amount of DNA coiling by adding negative supercoils or by unlinking DNA circles for prokaryotic DNA. Topoisomerase I relaxes the supercoil, and topoisomerase II adds negative supercoils. Topoisomerase is known as DNA gyrase in prokaryotes. Once the DNA strand is open, there needs to be a base for the DNA polymerase to bind and begin replication; this is provided by the primase. Primase adds short strands of RNA primers (9 to 12 pairs) onto the template to allow for DNA polymerase III or DNA polymerase alpha to bind and add nucleotides. DNA polymerase III (in prokaryotes) has 5’ => 3’ synthesis and 3’ => 5’ proofreading exonuclease. DNA polymerase alpha (in eukaryotes) is a complex that has the DNA primase which creates the RNA primer, and then the polymerase alpha itself elongates around 20 nucleotides and passes off to DNA polymerase epsilon or delta. DNA polymerase epsilon elongates and proofreads on the leading strand. DNA polymerase delta elongates and proofreads on the lagging strand. Because the strands run antiparallel, the polymerization mechanism is slightly different for both strands. The DNA polymerase runs in the 3’ => 5’ direction (therefore creating DNA in the 5’ => 3’ orientation), but only one DNA template strand, known as the leading strand, is in the proper orientation. Replication of the leading strand is simple because it is already 3’ => 5’ direction, the polymerase continuously adds complementary nucleotides to the primer towards the replication fork. For the lagging strand, which is 5’ => 3’, the new strand is synthesized in segments and discontinuously because the DNA polymerase can only read in the 3’ => 5’ direction. A new primer is added as the replication fork is further opened and the DNA polymerase delta builds the new DNA strand away from the replication fork, creating Okazaki fragments. Afterwards, another DNA polymerase replaces the RNA primers with DNA; this is done by DNA polymerase I in prokaryotes and DNA polymerase delta in eukaryotes. DNA ligase connects the fragments.

Termination

In prokaryotes, replication ends when the forks meet. In eukaryotes replication ends at telomere regions. Telomeres are regions at the end of chromosomes with repetitive nucleotides such as TTAGGG sequences. Shortening telomeres have been associated with cell aging and death.

DNA Proofreading

The DNA replication is not perfect and has mechanisms to ensure there are corrections. DNA polymerases make mistakes in 1 in 10^5 base pairs. Considering the length of the human genome is around 3 x 10^9 that is about 30,000 to 50,000 mistakes. However, with the exonuclease activity of the polymerases, the error rates are reduced to a few hundred. These errors are further corrected in the G2 phase of the cell cycle, reducing the total number of errors to less than 10.

Testing

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a method of rapidly amplifying DNA. It is used clinically to test for the presence of specific bacteria and viruses in patients with certain diseases. Nucleic acid amplification testing uses PCR and is used to diagnose chlamydia. Also, reverse transcriptase PCR is used to detect HIV.[7]

Southern Blot[8]

Southern blotting is done by separating DNA fragments through electrophoresis, and then transferred the DNA from the electrophoresis gel to a filter. The filter is then washed with radioactively labeled hybridization probes that target for the DNA sequence of interest. Once the probes bind, the filter is exposed to x-ray, and the probes marking the DNA of interest are visible. Southern blot can be useful clinically for detecting mutations in certain DNA sequences.

Clinical Significance

Antibiotics

Many antibiotics antivirals interfere with DNA replication in prokaryotes to prevent the replication of bacterias and viruses and the progression of disease in patients. One example includes fluoroquinolones, which bind to DNA topoisomerases and inhibit their function and preventing replication. Trimethoprim and sulfonamides prevent the formation of DNA precursors such as purines and pyrimidine, preventing DNA formation. Acyclovir acts as a guanosine analog which is monophosphorylated by HSV thymidine kinase; this phosphorylation creates a triphosphate which inhibits viral DNA polymerase by chain termination.

Antitumor Medications

Antitumor medications also may interfere with DNA replication. Cytarabine used to treat leukemia is a pyrimidine analog that inhibits DNA polymerase. 5-Fluorouracil, used in treating colon cancer, decreases the production of thymidine which is a nucleotide needed for DNA production. Etoposide is an antitumor medication that inhibits topoisomerase II; it is used to treat solid tumors, leukemias, and lymphomas. Irinotecan inhibits topoisomerase I.