DNA replication is used by cells to allow cell division, either in reproduction or in the duplication of new cells in multicellular or unicellular organisms. The complexity of DNA replication process requires the involvement of a great number of specific enzymes. DNA is present in cells as a double helix molecule and when this helix is unwinding, a newly synthesized strand emerges along with a parental strand. The DNA strand used to produce the complementary strand is referred to as the template strand, which is used to synthesize complementary strands of each parental strand. Thus, replication is a semi-conservative process.
The diagram below (left) shows a simple color coded depiction of this semi-conservative process.
The precursor of each new nucleotide in the DNA strand corresponds to a deoxynucleoside 5’-triphosphate. During replication, two terminal phosphates are removed and the internal phosphate is covalently bonded to the deoxyribose of the raising DNA strand. The nucleotide addition requires the presence of a free hydroxyl group, which is available only at the correct end, so the addition of the nucleotide phosphate group bonds with the 3’-hydroxyl(OH) of the previous nucleotide. The enzymes that catalyze the addition of deoxy ribonucleotides are denominated DNA polymerases. There are several types of these enzymes, each one with a specific role. All known DNA polymerases work in 5’ to 3’ direction, but none of them are able to start DNA synthesis alone. Since DNA polymerase can only add nucleotides to the 3’-OH, in order to start a new strand, it requires a primer.
The primer is a nucleic acid molecule in which the DNA polymerase can add a nucleotide to. Often, the primer is a small RNA fragment instead of DNA. When the double helix is unwinding at the beginning of replication, an enzyme of RNA polymerization (primase) synthesizes the RNA primer with 11-12 nucleotides, which is complementary to the DNA template strand. At the end of the RNA primer, there is a 3’-OH group in which the DNA polymerase adds the first deoxy ribonucleotide. Later, the RNA primer will be removed and replaced by DNA.
Before DNA polymerase synthesizes a new DNA strand, the existent DNA double helix needs to undergo an unwinding process to expose the template strand. The unpackaged region is where replication will start, and it is designated the replication fork. The DNA helicase enzyme is responsible unwinding and separating the DNA double helix strands in an ATP-dependent process, exposing a small region of a single strand. Helicase can move along the double helix structure right at the front of the replication fork. There are specific regions for replication to get started, also known as replication origins.
The replication process always happens in 5’ to 3’ direction, which means that a new nucleotide is added to the 3’-OH group of the raising DNA strand. For this reason, the strand being synthesized uses the 3’-5’ strand as a template, and we call it the leading strand (continuous strand). DNA synthesis occurs continuously since there is always a free 3’-OH group. On the other hand, in the newly synthesized strand using as template the 5’-3’ DNA strand, the DNA synthesis occurs in a discontinuous process. It does not have available a free 3’-OH for a nucleotide to be added. Thus, in the lagging strand, it is necessary for primase to synthesize the primer repeatedly to make available a free 3’-OH group. In the continuous strand only one primer is required at the beginning of DNA synthesis, but the lagging strand is synthesized in short fragments, also known as Okazaki fragments. These fragments are posteriorly fused, generating a continuous strand.
A complex of proteins including DNA polymerase attaches to the DNA strand at the replication fork, and slides along the DNA template strand. Two DNA polymerases and protein complexes are required for DNA replication (one for each strand) at the replication fork. After the synthesis of the leading strand and lagging strand, a DNA polymerase with exonuclease activity is necessary to remove the RNA primer and add complementary DNA nucleotides. The last phosphodiester ligation is done by a DNA ligase enzyme. Ligase enzyme joins the DNA cuts that contain a 5’-PO4 and an adjacent 3’-OH group.
Transcription is the synthesis of ribonucleic acid RNA using DNA as template. There are three significant chemical differences between RNA and DNA:
- The RNA contains the ribose sugar instead of deoxyribose.
- RNA has uracil replacing the thymine from DNA.
- Excluding some viruses, RNA is not found as a double strand.
The replacement of deoxyribose by ribose affects the chemical properties of the nucleic acid and generally enzymes that catalyze reactions in DNA do not have any action in RNA (and vice-versa). Meanwhile, the substitution of thymine by uracil does not affect the base pairing, since both thymine and uracil pair with adenine with the same efficiency.
All RNA molecules are the product of DNA transcription. They play a role at two different levels, genetic and functional. At the genetic level, the mRNA carries the genetic information from the genome to the ribosome. On the other hand, the rRNA play a functional and structural role in the ribosomes and the tRNA are responsible for transporting the amino acids for protein synthesis. Some RNA molecules, including rRNA, can have enzymatic activity.
The transcription of genetic information is done by an RNA-polymerase enzyme in a similar fashion as DNA-polymerase does in DNA replication. RNA polymerase catalyzes the formation of phosphodiester between ribonucleotides. This is driven at the expense of energy released by hydrolysis of two phosphate bonds from ribonucleotides. Similarly to DNA synthesis, RNA synthesis is performed in the 5’-3’ direction, with ribonucleotides being added to a free 3’-OH group from a previous ribonucleotide.
Unlike DNA polymerase, RNA polymerase is able to start new strands independently. Consequently, there is no need for a primer. In order to start RNA synthesis, it is necessary that RNA polymerase recognizes the DNA initiation sequences, also referred to as promoters. After RNA polymerase binds the promoter, transcription is allowed to start. In this process, the DNA double helix at the promoter region is unwound by RNA polymerase, exposing the DNA template strand. When a DNA region has two close promoters, with opposite directions, transcription takes place using both DNA strands as templates but in different directions. When the newly synthesized RNA strand dissociates from DNA, the unwinding DNA closes again back to its original double helix structure.
RNA polymerase uses DNA double strand (dsDNA) as template, however, only one of the strands is transcribed for each gene. Compared to DNA replication where all genomic DNA is replicated, in transcription only small DNA fragments are transcribed. Usually, these fragments correspond to one gene. This system allows the cell the possibility to transcribe different genes, at distinct frequencies depending on the cell's requirements.
Usually, the transcription process involves the transcription of genes required for the cell at that exact time, which means that it is critical that transcription is finished at the right spot. This termination process is controlled by specific sequences of nucleotides in the DNA template strand.
Unlike bacteria, a great number of transcripts from eukaryotes have introns (unnecessary regions for translation), which will require further RNA processing. This RNA processing is called splicing and occurs in the cell’s nucleus. The splicing involves a protein complex, the spliceosome, which is responsible for removing the introns from transcripts and joining the remaining sequences, referred to as exons.
Additionally, there are two other steps for mRNA processing in Eukaryotes. The first one is the capping process, which occurs before transcription termination. The capping process is the addition of a methylated guanine nucleotide cap at the 5’-end of the pre-mRNA. This cap will be crucial for translation initiation. The second step includes cleavage of transcript 3’-end, followed by addition of 100-200 adenylate residues, in a poly-A tail. This tail gives stability to the mRNA and its degradation will be required to allow RNA degradation.