Ch 14 DNA Replication and Repair 11 14 2014 11 14 2014 14 DNA Structure and Replication 11 14 2014 III DNA Replication A DNA replication is semiconservative Semiconservative half of a newly made DNA is the old template One strand is used as a template to make a new strand based on the complementary base pairing rules A T and G C 1953 Kornberg performed first in vitro DNA synthesis using a template DNA DNA polymerase and four nucleotides 1957 Meselson and Stahl proved the semiconservative mechanism They used 14N and 15N isotopes to distinguish the old DNA template and new DNA made from the template They grew bacteria in a medium containing 15N for 17 gen which resulted in nitrogenous bases in DNA labeled w 15N They collected bacterial samples from this culture and then inoculated into a medium cont 14N They allowed it to replicate once and collected the second sample The bacteria were allowed to replicate once more to undergo after one DNA replication another DNA replication 3rd sample was collected after 60 min They isolated DNA from these samples using cesium chloride density gradient centrifugation which can separate a soln acc to density of each DNA Results indicated that the new DNA strand synthesized in sample 2 after one DNA replication cell division is a combo of old and new DNA strands B Mechanism of DNA replication DNA replication starts at the origin of replication which is a location on DNA molecule w a specific sequence that is recognized by enzymes involved in DNA replication Bacterial chromosomes and plasmids contain a single origin of replication 245 bp long Eukaryotes have multiple origins and the length is not well defined DNA replication in the cell is complex and requires the concerted action of several proteins In vitro DNA template DNA primer Mg DNA polymerase dNTPS in a suitable buffer Topoisomerase I relaxes the supercoiled DNA Helicase unwinds the double helix into single stranded DNA SSB ssDNA binding protein stabilizes the ssDNA Primase synthesizes RNA primers DNA polymerase I erases RNA primers and fills in the gaps DNA polymerase III holoenzyme DNA replication proof reading and repair DNA ligase covalently joins free 3 and 5 ends of two DNA strands DNA gyrase introduces supercoiling in DNA DNA replication starts at the ORI and proceeds bidirectionally The replication bubble consists of two replication forks joined together which expand wider as the DNA replication progresses Initiation Supercoiling relaxed at the origin of replication ORI by topoisomerase single origin in prokaryotes and multiple in eukaryotes Relaxed DNA helix is open to make a replication fork by helicase Resulting ssDNA is stabilized by SSB proteins Primase makes an RNA primer providing a free 3 OH group for the DNA polymerase to use to start the new DNA synthesis Elongation DNA polymerase III a complex protein binds to the DNA template RNA primer and adds nucleotides complementary to the template strand 1000 nucleotides sec in prokaryotes New DNA synthesis occurs in the 5 to 3 direction on a template that runs in the 3 to 5 direction o This is due to the nature of the enzyme DNA polymerase which links new deoxy nucleoside triphosphates dNTPs to the 3 OH group of the growing strand o H bonding in the base pairing works only if the new strand runs in the opposite direction of the template DNA A leading strand is synthesized continuously from 5 to 3 based on the template Since the opposite strand is not open to continue DNA synthesis from 5 to 3 direction DNA is synthesized in small fragments 100 200 bases in eukaryotes and 1000 2000 in prokaryotes called Okazaki fragment Lagging strand made slowly after the leading strand Once the new DNA strand is synthesized DNA polymerase III proof reads it and removes errors DNA polymerase I removes the RNA primer and completes the DNA strand Once the small fragments are complete DNA ligase joins the two ends of DNA strands to complete DNA replication DNA gyrase facilitates supercoiling the DNA to compact the chromosomes into nucleosomes IV DNA repair Important for cells to correct errors in DNA replication or any damage caused to the DNA after replication bc it could be fatal Mistakes must be repaired before the DNA can function and replicate again If left uncorrected stable mutations result and may be passed on to the next gen of cells or organisms Three major types of DNA repair mechanism operating in the cell o Mismatch repair done to correct errors made during DNA replication DNA polymerase III makes 1 mistake in 10 000 base pairs It then proof reads the newly made DNA removes the wrong bases and repairs the DNA to minimize the errors to 1 in a bil Use the mismatch repair mechanism by checking complementary base pairing o Telomere repair or preservation occurs during DNA replication and is catalyzed by telomerase helps prevent the telomere from getting shortened after each cell cycle Telomerase temp extends the telomere region to allow the RNA primer to bind for DNA replication and once it is replication the extended region annealed to the RNA primer is removed to maintain the same length of telomere Telomerase is active in young organisms w actively growing cells and in cancer cells o Excision repair occurs after a cell divides and is in the G1 G2 phases This damage is caused by carcinogens and mutagenic radiations altering the bases or making pyrimidine dimer Such mutations are constantly monitored by 50 different enzymes are corrected by excising the damaged strand of DNA and making a new matching strand