DNA Replication PDF

# DNA Replication ## Replication Process - The replication process begins with the separation of DNA strands at various locations. - These locations are rich in A and T bases and are called "points of origin" or "consensus areas." - Bacterial DNA only has one point of origin. - The enzyme that sep...

# DNA Replication ## Replication Process - The replication process begins with the separation of DNA strands at various locations. - These locations are rich in A and T bases and are called "points of origin" or "consensus areas." - Bacterial DNA only has one point of origin. - The enzyme that separates DNA strands at the point of origin is called DNA-A protein. - Once this area is unzipped, it is called a replication bubble. - Single-Strand binding proteins (SSB proteins) bind to DNA strands to prevent them from reannealing and also protect them from being chopped up by endonucleases. - To further unwind the DNA spiral, an enzyme called Helicaze enters each replication fork site and separates the DNA strands downstream. - Helicaze requires a lot of energy to break multiple hydrogen bonds. - As Helicaze moves along and unzips DNA strands, tightly twisted zones of DNA downstream called supercoils are created. - A double-function enzyme called Topoizomerase is used to resolve these supercoils. ## Topoizomerase - Topoizomerase has two domains: a nuclease domain and a ligase domain. - The nuclease domain cuts only one strand of DNA in supercoiled areas, then the ligase domain of the Topoizomerase glues the broken chain back in the exact location it was cut. - There are different types of Topoizomerases. - Topoizomerase 2 has two nuclease domains and cuts both strands of DNA when different DNA molecules get tangled up during simultaneous replication. ## Antibiotics - Some antibiotics, such as Quinolones, work by deactivating the ligase domain and stimulating the nuclease domain of the Topoizomerase. - This results in DNA being chopped up but not repaired, which leads to the desintegration of DNA, ultimately killing bacteria. - Quinolones primarily work on bacterial Topoizomerase. ## Cancer Drugs - Cancer drugs like Etoposides and Teniposides work on this enzyme in humans, targeting cancer cells that are trying to replicate. ## Actual Replication - To start actual replication, an enzyme called Primase (DNA-dependent RNA polymerase) comes to the replication bubble and makes a small chain of RNA complementary to the DNA segment. - This small strand of RNA is called a Primer. - The primer provides a 3' end for DNA-dependent DNA polymerase to start building the DNA chain. - DNA polymerase has several domains and performs the following tasks: - Reads template DNA in the 3' to 5' direction. - Polymerizes the new strand in the 5' to 3' direction. - Proofreads the new strand in the 3' to 5' direction. - If it finds that the last nucleotide was an error, it uses its exonuclease domain to cut out that nucleotide. - It reads the last nucleotide on the DNA template again and puts a correct nucleotide in the new strand. - It proofreads again and, if no mistake was found, it moves on to the next nucleotide in the template DNA strand. ## Leading Strand - The new DNA strand that is synthesized off the primer in the direction of the replication fork is called the leading strand, and it is synthesized continuously. ## Lagging Strand - New DNA strand that is made off the primer in the direction away from the replication fork is called the lagging strand. - The lagging strand is made in shorter fragments off the new primers as the replication fork advances due to the DNA spiral unzipping. ## Primosome - To make these primers, enzyme Primase travels along the template strand of DNA in a complex with other proteins called Primosome. ## Okazaki Fragments - The shorter fragments of new DNA in the lagging strand from a primer to another primer are called Okazaki fragments. ## DNA Polymerase 1 - To deal with the problem of fragmented DNA on the lagging strand, another enzyme called DNA polymerase I is used. - It performs everything that DNA polymerase III does but, additionally, it has another exonuclease domain that removes RNA phosphodiester bonds from the 5' end of the primer. - DNA polymerase I starts chopping off nucleotides from the 5' end of the primer one by one, replacing them with DNA nucleotides. - Once all of the primer is replaced by DNA, then DNA Ligase comes in and stitches the ends of Okazaki fragments together. ## Pseudonucleotides - There is a class of drugs that are pseudo-nucleotides or preudonucleosides that have a modified deoxyribose, where the OH group is missing on carbon #3. - DNA polymerase I or III cannot recognize this altered sugar structure and incorporates these modified nucleotides into the DNA strands being synthesized, stopping further elongation of the DNA chain. - These drugs are usually antiviral or anticancer. Examples include: - Didinozine - antiviral, anti-HIV (analogue of Inosine) - Vidarabine - antiviral (analogue of Adenosine) - Acyclovire - antiviral (analogue of Guanine) - Cytarabine - anticancer (analogue of Cytosine) - Zidarative - antiviral (HIV) (analogue of Thymidine) ## Mitotic Spindle - There are two medications that affect the mitotic spindle of dividing cells: Vincristine and Vinblastine. - Both destroy protein tubulin, which is the building block of the mitotic spindle. - Vinblastine is more aggressive than Vincristine and causes ablation of bone marrow. ## Hyperstabilizing - Another way to attack the mitotic spindle is to hyperstabilize it and prevent it from shrinking and separating the chromatids. - Drugs that do this are called Paclitaxel and Docetaxel. ## Polymerases in Eukaryotes - The polymerases used in DNA replication are only found in prokaryotic cells. - Eukaryotic cells have a different set of polymerases: - **X-pol:** Acts as primer and makes very short DNA strands. - **B-pol:** DNA repair, corrects mismatched bases. - **Y-pol:** Used for the replication of mitochondrial DNA. - **δ-pol:** Elongates leading strand, makes Okazaki fragments. - **ε-pol:** Also concerned with DNA repair.