Transcript Slide 1
Bio3124 Lecture 8 DNA Contains Cell Information • Total cell DNA = genome (chromosome & extra-chromosomal) • Human genome = 4 billion bp – 1000x as large as E. coli genome – 90% junk DNA – ~8x more genes: 30,000 (human) vs. 4,000 (E. coli) • Bacterial genomes = 0.6–9.4 Mbp – Genome of bacteria usually circular • Seldom linear, segmented Bacterial Genetic Organization • E. coli genome – regulatory • promoter/operator, signal sequences – coding sequences – Average 1000 bases per bacterial gene – Organized on both strands – Operons and regulons – Monocistron vs Polycistron organization – Overlapping genes => ribosomal frameshifting Overlapping genes Met Pro Gln Pro Lys Trp Thr Lys Ile Cys Ser Leu His ATGCCCCAA---//---CCAAAATGAACGAAAATCTGTTCGCTTCAT Met Asn Glu Asn Leu Phe Ala Ser DNA is an antiparallel double helix • Geometry of bases and their spacial arrangement to form H-bond cause helix structure of dsDNA Major groove • B-form DNA • pairing bases stack at the centre • backbone intertwined • creates minor and major grooves 34 A • 0.34 nm (3.4 A) rise per base pair • one full helix turn houses 10 nucleotides 20 A DNA is an antiparallel double helix Major groove 34 A 20 A 6 DNA Is Packed to Fit the Cell DNA Is Packed to Fit the Cell Nucleoid of E. coli Circle of dsDNA 1500x the size of the cell • Multiple loops held by anchoring proteins • Each loop has coiled DNA Supercoiling Compacts DNA • Unsupercoiled DNA = 1 winding for 10 bases • Positive supercoils – Winding more frequently • Overwinding • Negative supercoils – Winding less frequently • Underwinding • Supercoils twist DNA • Why supercoils are important? – Eubacteria => less frequent winding – Extreme thermophiles => more frequent winding Relevance to Research 1 2 3 Circular Linear Super-coiled Topoisomerases Regulate Supercoils • Type I Topoisomerases – Relieve torsional stress caused by supercoils – Act on one strand, How? • Type II Topoisomerases (DNA gyrase) – Unwind dsDNA – Introduce negative supercoils – Act on both strands of dsDNA, How? • Archaeal topoisomerases – Reverse topoisomerases – Introduce positive supercoils Topoisomerase I • Single protein, nicks one strand • Allows passages of the other strand through single strand break • Releaves accumulated positive supercoils ahead of replicating DNA Topoisomerase II (DNA Gyrase) • two subunits, GyrB and GyrA • GyrB binds DNA, passes to GyrA • GyrA introduces double strand break – 2 ATP hydrolysed – Remains transiently attached • Passes other dsDNA through break • Reseals the ds break • A negative writhe introduced Mechanochemical analysis of DNA gyrase Topoisomerases Regulate Supercoils Summary Animation: Topoisomerases I and II DNA Replication Semiconservative replication • Copies information from one strand to a new, complementary strand – Dividing cells receive one parental strand and one newly synthesized strand – Melt double-stranded DNA – Polymerize new strand complementary to each melted single strand Replication Begins at oriC oriC ter ‘13-mers’ ‘9-mers’ E. coli oriC: 245 bp Replication Begins at oriC • Timing: Dam methylation at A of GATC (ie. GAN6mTC) • SeqA binds to hemi methylated duplex at OriC • Full methylation following cell division and loss of SeqA affinity • DnaA concentration rises • Binds to 9-mer repeats at OriC OriC: 245 bp contining 9-mer repeats, with 13-mer repeats in between DnaA binding, strand melting at 13-mer by RNAP DNA Helicase Melts DNA • Helicase Loader (DnaC) places helicase (DnaB) at each end of origin Helicase Loader Origin Helicase Recruits Primase • Primase begins replication • RNA primer forms 3OH for DNA to attach – Evolutionary remnant? – 1st cells thought to use RNA, not DNA Helicase Primase Primosome Primer Recruits Clamp Loader to Each Strand • Sliding clamp binds DNA polymerase III to each strand DNA Pol III Sliding Clamp Clamp Loader DNA Pol III Polymerase Proceeds 5 3 on Each Strand • Energy for polymerization comes from phosphate groups on added base. – Must add new base to 3OH of a chain – New nucleic acids grow to extend 3 end Each Fork Has Two Strands • Steady growth of new “leading” strand – Leading strand follows helicase • Lagging strand: discontinuous, needs intermittent release and reloading of replisome Leading Strand Leading Strand Lagging Strand Lagging Strand Growth • Polymerase continues to previous primer • Clamp loader places primase on new site • DNA present in 1000 base pieces – Okazaki fragments RNase H Removes Primers • One primer for each leading strand • Many primers on lagging strands – One per Okazaki fragment • Gaps filled in by DNA Polymerase I • Ligase seals nicks DNA Replication: Sliding model • Replisome anchored to membrane at mid-cell • DNA spools through as replicated • Proof? • PolC-GFP stays at equator attached to membrane • DAPI stained DNA: throughout cytoplasm Animation: Summary of DNA Replication Relevance to Research • DNA replication in vitro • Polymerase chain reaction (PCR) – Amplifies specific genes from a given genome – Need: template DNA, primers, dNTPs, DNA Polymerase, buffer, Mg2+ fd – Denaturation, Annealing, Elongation PCR cycles 10 20 30 40 Animation: Proof reading function of Pol III Both Forks Move to ter Sites • Movement is simultaneous • Opposite directions until both meet again at terminus • Replisome disassembles at ter sites Plasmids • Extrachromosomal pieces of DNA • Low-copy-number plasmids – One or two copies per cell – Segregate similarly to chromosome • High-copy-number plasmids – Up to 700 copies per cell – Divide continuously – Randomly segregate to daughter cells Plasmid Genes • Advantageous under special conditions – Antibiotic-resistance genes – Genes encoding resistance to toxic metals – Genes encoding proteins to metabolize rare food sources – Virulence genes to allow pathogenesis – Genes to allow symbiosis Relevance to Research • Molecular cloning – Plasmids are used to import a segment of exogenous DNA into a host cell. Plasmid Replication Bidirectional replication Similar to chromosomal replication Unidirectional (“rolling circle”) replication Starts at nick bound by RepA protein Provides 3OH for replication Helicase moves around plasmid repeatedly Complementary strand synthesized Used by many bacteriophages Animation: Rolling circle replication