1. Bio3124 Lecture 8

2. 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 DNA Contains Cell Information

3. 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

4. ATGCCCCAA---//---CCAAAATGAACGAAAATCTGTTCGCTTCAT MetProGlnProLysTrpThrLysIleCysSerLeuHis MetAsnGluAsn LeuPheAlaSer Overlapping genes

5. DNA is an antiparallel double helix Geometry of bases and their spacial arrangement to form H-bond cause helix structure of dsDNA B-form DNA pairing bases stack at the centre backbone intertwined creates minor and major grooves 0.34 nm (3.4 A) rise per base pair one full helix turn houses 10 nucleotides 34 A Major groove 20 A

6. DNA is an antiparallel double helix 34 A Major groove 20 A

7. DNA Is Packed to Fit the Cell 6

8. Multiple loops held by anchoring proteins Each loop has coiled DNA Nucleoid of E. coli Circle of dsDNA 1500x the size of the cell

9. 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

10. Circular Linear Super-coiled Ladder 1 2 3 Relevance to Research

11. 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

12. 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

13. 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

14. Topoisomerases Regulate Supercoils

15. Summary Animation: Topoisomerases I and II

16. 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

17. oriC ter ‘9-mers’ ‘13-mers’ Replication Begins at oriC E. coli oriC: 245 bp

18. Replication Begins at oriC Timing: Dam methylation at A of GATC (ie. GA N6m TC) 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

19. DNA Helicase Melts DNA Helicase Loader (DnaC) places helicase (DnaB) at each end of origin Helicase Origin Loader

20. 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

21. Primer Recruits Clamp Loader to Each Strand Sliding clamp binds DNA polymerase III to each strand DNA Pol III Sliding Clamp Clamp Loader

22. 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

23. 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

24. Lagging Strand Growth Polymerase continues to previous primer Clamp loader places primase on new site DNA present in 1000 base pieces – Okazaki fragments

25. 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

26. 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

27. Animation: Summary of DNA Replication

28. 10 20 30 40 PCR cycles DNA replication in vitro Polymerase chain reaction (PCR) – Amplifies specific genes from a given genome – Need: template DNA, primers, dNTPs, DNA Polymerase, buffer, Mg 2+ fd – Denaturation, Annealing, Elongation Relevance to Research

29. Animation: Proof reading function of Pol III

30. Both Forks Move to ter Sites Movement is simultaneous Opposite directions until both meet again at terminus Replisome disassembles at ter sites

31. 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

32. 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

33. Relevance to Research Molecular cloning – Plasmids are used to import a segment of exogenous DNA into a host cell.

34. 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

35. Animation: Rolling circle replication