GEN 3022 1st Edition Lecture 16 Outline of Last Lecture I Structure of bacterial chromosomes a Location b Overall shape structure i Loop domains ii DNA supercoiling II Organization of Eukaryotic chromosomes a Chromosome replication and segregation i Three types of DNA sequences b Compaction of DNA i Process interphase and metaphase ii Heterochromatin iii Euchromatin III Nucleosomes a Definition b Components i Histone proteins ii DNA Outline of Current Lecture I Overview of DNA replication These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute II III IV V VI VII VIII IX a Replication patterns i Antiparallel ii Chargraff s rule iii Semiconservative b Summary Proposed models of DNA replication a Conservative model b Semiconservative model c Dispersive model Experiment a E coli growth b Light and half heavy DNA types Origin of bacterial DNA replication a Origin of replication oriC b Patterns of bacterial DNA replication Synthesis of new DNA strands a DNA helicase b Topoisomerase II DNA gyrase c Primase d DNA Polymerases e Ligase Synthesis a Leading strands b Lagging strands c DNA polymerase I action d DNA ligase action Fidelity mechanisms a High fidelity in DNA replications i Three reasons b Proofreading activity of DNA polymerase Eukaryotic genomes Telomeres and DNA replication a Telomeric sequences b Telomerase c Telomere length and cancer Current Lecture I Overview of DNA replication a Replication patterns i Antiparallel one strand runs 5 to 3 and the other 3 to 5 ii Chargraff s rule A T C G iii Semiconservative one parent strand and one daughter strand II III IV V VI b Summary i Two complementary strands of DNA separate ii Each serves as a template strand for the synthesis of new complementary daughter DNA strands Proposed models of DNA replication a Conservative model both parental strands stay together after DNA replication b Semiconservative model the double stranded DNA contains one parental and one daughter strand following replication c Dispersive model parental and daughter DNA segments are interspersed in both strands following replication Experiment a E coli growth Meselson and Franklin Stahl investigated DNA replication using E coli i E coli was grown in the presence of a heavy isotope of nitrogen so that the population of cells all had heavy labeled DNA ii E coli was switched to a medium with only light isotope of nitrogen iii Density of resulting generations was analyzed to determine the pattern of replication b Light and half heavy DNA types i The first generation s DNA types were consistent with the dispersive model and semiconservative model but the second generation s types were only consistent with the semi conservative model Origin of bacterial DNA replication a Origin of replication oriC since bacterial chromosomes are circular there is only one origin of replication called the origin of Chromosomal replication oriC b Patterns of bacterial DNA replication synthesis of DNA proceeds bidirectionally around the chromosome until the replication forks meet and replication is terminated Synthesis of new DNA strands a DNA helicase responsible for separating the two template strands into a replication fork b Topoisomerase II DNA gyrase unwinds negative supercoiling of DNA so that it can be separated for replication c Primase creates RNA primer segments that covalently link to the template strand and are later removed by DNA polymerase I d DNA Polymerases responsible for DNA synthesis work in the 5 to 3 direction only and cannot initiate transcription DNA polymerase III is responsible for the most synthesis e Ligase covalently links Okazaki fragments of lagging strand by catalyzing formation of the phosphodiester bond Synthesis VII VIII IX a Leading strand strand that gets synthesized continuously using one RNA primer from 5 to 3 towards replication fork b Lagging strand strand that gets synthesized in fragments from 5 to 3 away from the replication fork c DNA polymerase I action removes RNA primer and fills in the gaps with newly synthesized DNA d DNA ligase action covalently links Okazaki fragments after DNA polymerase I fills in the gaps that are created by the removal of RNA primer Fidelity mechanisms a High fidelity in DNA replications mistakes during DNA replication are extremely rare DNA polymerase III makes only one mistake per every 108 bases made i Three reasons stability of base pairing structure of DNA polymerase active site proofreading function of DNA polymerase b Proofreading activity of DNA polymerase DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand The enzyme uses a 3 to 5 exonuclease activity to digest the newly made strand until the mismatched nucleotide is removed Eukaryotic genomes a Unlike bacterial genomes eukaryotic chromosomes are long and linear as opposed to circular b As a result they also have many origins of replication about every 100 000 base pairs Telomeres and DNA replication a Telomeric sequences i Sequences at the end of a chromosome that codes for the stop of replication ii Typically consist of moderately repetitive tandem arrays with a 3 overhang that is 12 16 nucleotides in length These nucleotides are generally many guanine and thymine b Telomerase i Since DNA polymerases can only work in the 5 to 3 direction and cannot initiate DNA synthesis a special enzyme telomerase is needed to complete replication ii There is not enough DNA at the end of a strand to allow for RNA primer to attach which prevents DNA replication from completion iii Telomerase builds on to the end of a DNA strand to allow for RNA primer to bind and for replication to finish c Telomere length and cancer i Telomere DNa is about 8 000 at birth and can shorten to 1 500 bp in an elderly person ii Telomeres shorten in actively dividing cells which makes some cells senescent loss of ability to divide Insertion of highly active telomeres can block senescence iii Cancer cells commonly carry mutations increasing activity of telomerase which prevents telomere shortening and senescence May be a target for anti cancer drug treatments