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Spring 09 lecture 10 and 11

Spring 09 lecture 10 and 11 - Before we start to talk about...

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11 Before we start to talk about DNA replication… Specific proteins recognize specific sites on NA and bind; DNA (RNA) – protein interactions – very important Enzymatic: nucleic acids – substrates, protein - enzymes Ø DNA: restriction enzymes, topoisomerases, telomerases, methylases, polymerases etc. Ø RNA: tRNA synthetases, polymerases, polyadenylases Structural: change in DNA/RNA structure Ø helicases (unwinding parental DNA duplex during DNA replication), histones, TBP etc. Regulatory Ø In both transcription (DNA binding) and translation (RNA binding)
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22 Types of protein - NA interactions : • + charge to -charge • Hydrogen bonds • Van der Waals • Hydrophobic interactions Keep in mind: • DNA and RNA are negatively charged - binding proteins tend to be positively charged (e.g. histone) • DNA and RNA are surrounded by positively charged ions - when protein binds to nucleic acid, positively charged ions are displaced (energy requirement etc.) Major (and minor) groove
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33 DNA replication Learning Objectives Semiconservative and bidirectional Describe the cis elements and trans factors involved in the process of replication initiation Describe how ori sequences are located and studied Initiation of replication
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44 Replication is semiconservative Efficient process - it always starts at the defined sequence of base pairs Replication origin : a stretch of DNA necessary and sufficient for beginning of replication bacterial and plasmid genomes often have only one (more complicated in eukaryotic cell – multiple origins ) Each region served by one DNA origin is replicon - any piece of DNA which replicates as a single unit Bidirectional from the origin in majority of organisms: Replication fork Replication bubble Leading and lagging strands DNA Replication
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55 Cultured mamalian cells High and low concentrations of 3H-T DNA labeled near replication origin DNA dried on a microscope slide Autoradiographic patterns
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66 How were the enzymes involved in DNA replication discovered? DNA replication first studied in prokaryotes using both genetic and biochemical approaches: Make a mutant population and screen for mutants affected in DNA replication Characterize as to whether replication stops quickly or slowly under non-permissive conditions Purify enzymes required for replication in vitro Assay : Add radiochemical dNTP to an extract containing enzymes necessary for replication, along with a template DNA and measure incorporation of radioactivity into new DNA (can be precipitated and collected on a filter)
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77 DNA replication machinery Several enzymes and other factors involved in DNA replication (10 major enzymes/proteins to know) DnaA -initiation DnaB - helicase and DnaC - “clamp loader” Single-strand binding proteins ( SSB ) Primase (DnaG) RNase H DNA polymerase ( I and III ) DNA ligase Topoisomerase
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88 Replisome : combination of all the proteins that function at the replication fork, & undertake the synthesis of DNA (Lewin, 2006.
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