BIOL 3451 1st Edition Lecture 13 Outline of Last Lecture I 10 10 Many Analytical Techniques Have Been Useful during the Investigation of DNA and RNA II 11 1 DNA is Reproduced by Semiconservative Replication III 11 2 DNA Synthesis in Bacteria Involves Five Polymerases as well as Other Enzymes IV 11 3 Many Complex Issues Must Be Resolved during DNA Replication Outline of Current Lecture I 11 3 Many Complex Issues Must Be Resolved during DNA Replication II 11 4 A Coherent Model Summarizes DNA Replication III 11 5 Replication Is Controlled by a Variety of Genes IV 11 6 Eukaryotic DNA Replication Is Similar to Replication in Prokaryotes but Is More Complex V 11 7 The Ends of Linear Chromosome Are Problematic during Replication VI 11 8 DNA Recombination Like DNA Replication Is Directed by Specific Enzymes VII 12 1 Viral and Bacterial Chromosomes Are Relatively Simple DNA Molecules VIII 12 2 Supercoiling Facilitates Compaction of the DNA of Viral and Bacterial Chromosomes IX 12 3 Specialized Chromosomes Reveal Variations in the Organization of DNA Current Lecture I 11 3 Many Complex Issues Must Be Resolved during DNA Replication DNA polymerase I chop out and put one back behind it Knocks out RNA to replace with DNA DNA ligase DNA strand Figure 11 12 Beta subunit clamp prevents core enzyme from falling off template during DNA synthesis Proofreading and error correction important for DNA replication System of enzymes that looks for all the base pairs to be correct if mismatched cuts out one on new strand and have another enzyme come in and place correct pair there 3 to 5 exonuclease activity that allows proofreading II 11 4 A Coherent Model Summarizes DNA Replication Dna A Enzymes to know Figure 11 13 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 III IV Helicase opens up helix ATP Gyrase removes any positive supercoiling ATP keeps negative supercoiling Protein SSB to keep open Primase lay down primers rNTPs DNA polymerase III replicase dNTPs DNA polymerase III dNTPs 7 DNA ligase NADT DNA polymerase I knocks out primers in different locations 11 5 Replication Is Controlled by a Variety of Genes Once begin replicating chromosome you commit to it Some can do multiple origins at one chromosome Conditional mutations Table 11 4 Temperature sensitive mutation may not be expressed in a particular permissive temperature but when mutant cells expressed in restrictive temp mutant phenotype can be expressed 11 6 Eukaryotic DNA Replication Is Similar to Replication in Prokaryotes but Is More Complex Similarities between eukaryotic DNA replication and bacteria replication Double stranded DNA unwound at replication origins Replication forks formed Bidirectional synthesis has leading and lagging strands Eukaryotic polymerases require four deoxyribonucleoside triphosphates a template and a primer Eukaryotic DNA replication is more complex than bacteria because More DNA than prokaryotic cells Chromosomes are linear with many origins on one chromosome DNA is complexed with proteins everything done in context of chromatin 1 Where more replicons come in origins of replication One chromosome many origins many replicons Allows genome to be replicated in a matter of minutes to a few hours Figure 11 14 note all of the origins 1 Fire by region not all at same time therefore not random usually in groups Yeast about 10 million genes more complex than bacteria Has about 250 400 origins Called autonomously replicating sequences ARSs Eukaryotic origins also control timing of DNA replication Prereplication complex pre Rc assembles and replicates origin 1 Origin recognition complex ORC six protein complex that recognizes replication origins and tags the them as the sites of initiation in early G1 phase of cell cycle V VI Three DNA polymerase involved in replication of nuclear DNA One involves mitochondrial DNA replication Others involved in repair processes Table 11 5 Polymerases alpha delta and epsilon are major forms of the enzyme involved in initiation and elongation Polymerase alpha possesses low processivity reflects length of DNA that is synthesized by an enzyme before it dissociates from the template Can include synthesis of RNA primers during initiation on leading and lagging strands Once primer in place polymerase switching occurs polymerase alpha and epsilon are replaced by polymerase delta for elongation Polymerase delta synthesizes lagging strand and polymerase epsilon synthesizes leading strand 11 7 The Ends of Linear Chromosome Are Problematic during Replication When adding primers to linear DNA chromosome will get shorter and shorter so Telomere repeated sequence at end of chromosomes and preserve integrity and stability of chromosomes because it doesn t encode for any proteins what happens when run out of these Telomerease brings own RNA template and adds telomeres with aging you run out of telomeres and telomerase begins to stop production so you die eventually 1 Cancer cells turn the telomerase back on even when regular cells would not do that so cancer can kill you 2 Directs synthesis of telomere repeat sequence to fill gap 3 Enzyme is ribonucleoprotein with an RNA that serves as template for the synthesis of its DNA complement Reverse transcriptase a Figure 11 17 Particular problem with lagging strand because once RNA primer is removed there is no 3 hydroxyl group from which to elongate Fig 11 16 Most eukaryotic somatic cells telomerase is not active Active in bone marrow where red blood cells made or stem cells it is active As division continues telomeres shorten and erode causing further division to stop Malignant cancer cells maintain telomerase activity and are immortalized a lot of study done on this 11 8 DNA Recombination Like DNA Replication Is Directed by Specific Enzymes Genetic recombination involves Endonuclease nicking Strand displacement and pairing with complement VII VIII Ligation Branch migration unwind both duplexes and then wind them back with each other Duplex separation to generate the characteristic Holliday structure Fig 11 18 1 His structure not completely true but close Genetic exchange occurs at equivalent positions along two chromosomes with substantial DNA sequence homology is referred to as general homologous or recombination Not exactly right because there is a little bit of synthesis at beginning of recombination RecA protein in E coli promotes exchange of