Bio3124 Lecture 8

1. Bio3124Lecture 8 Bacterial Genetics:I. Genome replication and packing

2. 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

3. 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 Bacterial Genetic Organization

4. 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

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 Major groove 34 A 20 A

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

7. 6 DNA Is Packed to Fit the Cell

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

9. 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 Supercoiling Compacts DNA

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

11. 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? Archaealtopoisomerases Reverse topoisomerases Introduce positive supercoils Topoisomerases Regulate 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. 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 Topoisomerase II (DNA Gyrase) Mechanochemical analysis of DNA gyrase

14. Topoisomerases Regulate Supercoils

15. Summary Animation: Topoisomerases I and II

16. Semiconservativereplication 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 DNA Replication

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

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

20. Primase begins replication RNA primer forms 3OH for DNA to attach Evolutionary remnant? 1st cells thought to use RNA, not DNA Helicase Recruits Primase Helicase Primase Primosome

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

22. Energy for polymerization comes from phosphate groups on added base. Must add new base to 3OH of a chain New nucleic acids grow to extend 3 end Polymerase Proceeds 5 3 on Each Strand

23. Steady growth of new “leading” strand Leading strand follows helicase Lagging strand: discontinuous, needs intermittent release and reloading of replisome Each Fork Has Two Strands Leading Strand Leading Strand Lagging Strand

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

25. 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 RNase H Removes Primers

26. 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 DNA Replication: Sliding model

27. Animation: Summary of DNA Replication

28. 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

29. Animation: Proof reading function of Pol III

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

31. 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 Plasmids

32. 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 Plasmid Genes

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 3OH for replication • Helicase moves around plasmid repeatedly • Complementary strand synthesized • Used by many bacteriophages

35. Animation: Rolling circle replication