DNA replication is a semi-conservative process that occurs in the cytosol of prokaryotes and the nucleus of eukaryotes. It starts with the binding of the pre-priming complex at the origin of replication. Helicase unwinds the double helix, while single-stranded binding proteins protect the unwound DNA strands. DNA topoisomerases work to remove any supercoils created during the unwinding process. Following this, primase generates an RNA primer before DNA polymerase III starts to synthesize the new complementary DNA strand. Additionally, DNA polymerase III also removes mismatched bases through its 3 to 5 prime exonuclease activity.
In eukaryotic cells, telomerase adds a repetitive, non-coding sequence to the ends of chromosomes to prevent DNA degradation. High telomerase activity is associated with cancer, while shortened telomere lengths may suggest cellular senescence and aging. There are multiple types of drugs that block DNA replication, including topoisomerase inhibitors and chain elongation inhibitors, such as nucleoside analogs.
<ul> <li>DNA Replication</li> <ul> <li>Semi-conservative process</li> <li>Prokaryotes: replication in cytosol</li> <li>Eukaryotes: replication in nucleus</li> <li>Magnesium as a cofactor for enzyme function</li> </ul> <li>Prokaryotic DNA Replication</li> <ul> <li>Origin of replication</li> <li>Prepriming complex</li> <ul> <li>Helicase and associated proteins</li> <li>Binding to AT repetitive segments</li> <li>Partially separating DNA strands</li> </ul> <li>Helicase unwinds the double helix</li> <li>Single stranded binding proteins</li> <li>Positive and negative supercoils</li> <ul> <li>DNA topoisomerases</li> <li>Type 1 topoisomerase</li> <li>Type II topoisomerase (e.g. gyrase)</li> <li>Fluoroquinolone antibiotics target gyrase and topoisomerase 4</li> </ul> <li>RNA primers and primase</li> <li>DNA polymerase III</li> <ul> <li>Highly-processive enzyme</li> <li>Sliding clamp for enzyme stability</li> <li>Proofreading capabilities (3 to 5' exonuclease)</li> </ul> <li>DNA polymerase I</li> <ul> <li>5 to 3 prime exonuclease activity (removes RNA primer)</li> </ul> <li>Leading and lagging strands</li> <ul> <li>Okazaki fragments on lagging strand</li> <li>DNA ligase connects fragments</li> </ul> <li>Termination proteins</li> </ul> <li>Eukaryotic DNA Replication</li> <ul> <li>Multiple origin of replication sites</li> <li>More proteins required for replication</li> <li>Major proteins and their roles (table)</li> <li>Five different DNA polymerases identified</li> <li>Additional step: telomerase</li> <ul> <li>Non-coding, repetitive sequence added to chromosome ends</li> <li>Telomere shortening in successive replication rounds</li> <li>Cellular senescence and aging associated with shortened telomeres</li> <li>High telomerase activity in cancer cells</li> </ul> <li>Bloom syndrome</li> <ul> <li>Genetic disorder affecting helicase</li> </ul> <li>Inhibitors of DNA replication</li> <ul> <li>Topoisomerase inhibitors (e.g. irinotecan, topotecan, etoposide, teniposide)</li> <li>Chain elongation inhibitors (e.g. cytarabine, zidovudine)</li> </ul> </ul> </ul>
Prokaryotic DNA replication occurs in a circular DNA molecule, with a single origin of replication and proceeds bidirectionally, while eukaryotic DNA replication occurs in linear DNA molecules, with multiple origins of replication and proceeds simultaneously at various points. Moreover, eukaryotic DNA replication involves more complex machinery and a greater variety of DNA polymerases involved in the process.
DNA replication is semi-conservative because, during replication, each of the two parental DNA strands serves as a template for the synthesis of a new complementary daughter strand. As a result, each newly synthesized double helix contains one original parental strand and one newly synthesized strand. This ensures that the genetic information is conserved and passed down to the daughter cells.
DNA polymerase is an enzyme that synthesizes new DNA strands complementary to the template strands by adding nucleotides to the growing chain. Telomerase is an enzyme that catalyzes the addition of telomere sequences to the ends of linear eukaryotic chromosomes, preventing loss of genetic information during replication. DNA ligase is an enzyme that links Okazaki fragments together by catalyzing the formation of phosphodiester bonds, thereby creating a continuous DNA strand.
Okazaki fragments are short, newly synthesized DNA fragments on the lagging strand during DNA replication. As DNA replication is semi-conservative and occurs in a 5' to 3' direction, the lagging strand is replicated discontinuously in the form of Okazaki fragments, formed in the 5' to 3' direction away from the replication fork. These fragments are synthesized using short RNA primers and later joined together by DNA ligase to form a continuous strand.
Topoisomerase inhibitors block the action of topoisomerases, enzymes that alter DNA supercoiling and are essential for resolving the topological constraints in DNA replication. Blocking topoisomerases can lead to DNA breakage and the cessation of replication. Chain elongation inhibitors, on the other hand, disrupt the process of DNA strand elongation by interfering with DNA polymerase function or incorporating themselves into DNA strands. This leads to the termination of DNA synthesis, and consequently, halts DNA replication.