1. 1 Chapter 7 Genome and DNA Replication

2. 2 Introduction A genome is all the genetic information that defines an organism. Microbial genomes consist of one (usually) or more DNA chromosomes. This chapter explores the structure of genomes and their replication.

3. 3 Life is specified by genomes. Every organism, including humans, has a genome that contains all of the biological instructions needed to build and maintain a living example of that organism. The biological information contained in a genome is encoded in its DNA and is divided into discrete units called genes. Genes code for proteins, rRNA, tRNA or small RNAs

4. 4 In 1909, Danish botanist Wilhelm Johanssen coined the word gene for the hereditary unit found on a chromosome. Nearly 50 years earlier, Gregor Mendel had characterized hereditary units as factors— observable differences that were passed from parent to offspring. Today we know that a single gene (or set of genes) provides the complete instructions to make a functional product, called a protein. Genes instruct each cell type— such as skin, brain, and liver—to make discrete sets of proteins at just the right times, and it is through this specificity that unique organisms arise.

5. 5 Two types of gene transfer are known: - Vertical transmission: From parent to child - Horizontal transmission: Bacteria seem to operate differently where transfer of small pieces of DNA from one cell to another cell of same species or different species occurred frequently. Fred Griffith’s work on horizontal gene transfer, transformation led to the discovery that DNA is the genetic material. DNA: The Genetic Material

6. 6 Transformation Figure 8.24

7. 7 In 1944, Avery, Macleod, and McCarty discovered that the component responsible for transforming harmless strains of Steptococcus to a virulent strain was DNA. These results provided one of the conclusive indications that DNA was indeed the carrier of genetic material

8. 8 Genome organization

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10. 10 Types of genes Types of genes: regulatory and Structural (transcribed or functional) Structural gene: produces a functional RNA (tRNA,rRNA, mRNA, small RNAs). Only mRNA encodes a protein. Regulatory gene: regulates the expression of a structural gene. - Does not encode an RNA

11. 11 A gene can operate independently of others. - Or, it may exist in tandem with other genes in a unit called an operon. Functional Units of Genes

12. 12 DNA Structure

13. 13 DNA is a polymer of nucleotides. Each nucleotide consists of three parts: - Nitrogenous base - Purine: Adenine (A) and guanine (G) - Pyrimidine: Cytosine (C) and thymine (T) - Deoxyribose sugar - Phosphate Nucleotides are connected to each other by 5´-3´ phosphodiester bonds. Nucleosides and Nucleotides

14. 14 Bases in DNA: Adenine (A), Guanine (G), Cytosine (C) and Thymine (T) A Nucleoside: A base + Deoxyribose (a sugar) A Nucleotide: A base + Deoxyribose + Phosphate DNA structure elements

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16. 16 Organelle DNA Not all genetic information is found in nuclear DNA. Both plants and animals have an organelle—a "little organ" within the cell— called the mitochondrion. Each mitochondrion has its own set of genes. Plants also have a second organelle, the chloroplast, which also has its own DNA.

17. 17 Hydrogen bonding allows complementary base interactions. - A pairs only with T (via two H bonds). - G pairs only with C (via three H bonds). These interactions allow the two phosphodiester backbones to come together in an antiparallel fashion. - Thus forming the double helix DNA Structure

18. 18 5’ 3’

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21. 21 The DNA double helix has grooves: a wide major groove and a narrow minor groove. - These provide DNA- binding proteins access to base sequences. DNA Structure

22. 22 RNA differs from DNA: - Usually single-stranded - Contains ribose sugar - Uracil replaces thymine RNA Structure Figure 7.4B

23. 23 Bacteria pack their DNA into a series of loops or domains, collectively called the nucleoid. - Loops are anchored by histone-like proteins The Bacterial Nucleoid

24. 24 But how does DNA achieve this supercoiled state? The Bacterial Nucleoid Figure 7.9

25. 25 Positive supercoils: DNA is overwound. Negative supercoils: DNA is underwound. Eukaryotes, bacteria, and most archaea possess negatively supercoiled DNA. Archaea living in acid at high temperature possess positively supercoiled DNA. Enzymes that change DNA supercoiling are called topoisomerases. DNA Supercoiling

26. 26 Removal of one turn induces structural strain that can be accommodated by (c) strand separation over 10.5 base pairs or by (d) formation of a supercoil. Supercoiling induced by separating the strands of a helical structure. 'Iwist two linear strands of rubber band into a right- handed doublehelix as shown. Fix the left end by having a friend hold onto it. If the two strands are pulled apart at the right end, the resulting strain will produce supercoiling as shown.

27. 27 Type I topoisomerases - Usually single proteins - Cleave one strand of DNA - Relieve or unwind super coil Type II topoisomerases - Have multiple subunits - Cleave both strands of DNA - Introduce additional turns or coils - Example: DNA gyrase - Targeted by quinolone antibiotics Topoisomerases

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30. 30 DNA Replication Extraordinarily important and complex process. In bacteria, replication occurs at 750-1000 base-pairs/ second. E.Coli makes errors with a frequency of 10 -9 - 10 -10 base-pair replicated.

31. 31 Genetic Information flow Information Flows from DNA to RNA to Proteins in 3 processes 1.Replication (DNA  2 DNAs) 2.Transcription (DNA mRNA, rRNA, or tRNA 3.Translation (mRNA  Protein) DNA Replication

32. 32 Central dogma of molecular biology One way transfer of information from nucleic acid to protein is universal and holds for all forms of life on the planet

33. 33 DNA Replication Replication of cellular DNA in most cases is semiconservative. - Each daughter cell receives one parental and one newly synthesized strand.

34. 34 DNA Replication process Replication in bacteria begins at a single origin (oriC). After initiation, a replication bubble forms. -DNA methylation controls the timing of replication - Contains two replication forks that move in opposite directions around the chromosome Replication ends at defined termination (ter) sites located opposite to the origin.

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36. 36 Replication Fork

37. 37 Replication Machinery (replisome) The major proteins involved in DNA replication include: - DnaA: Initiator protein - DnaB: Helicase - DNA primase: Synthesis of RNA primer - DNA pol III: Major replication enzyme - DNA pol I: Replaces RNA primer with DNA - DNA gyrase: Relieves DNA supercoiling

38. 38 Initiation of Replication The start of DNA replication is precisely timed and linked to the ratio of DNA to cell mass. In E. coli, DnaA accumulates during growth, and then triggers the initiation of replication. - DnaA-ATP complexes bind to 9-bp repeats upstream of the origin. - This binding causes DNA to loop in preparation for being melted open by the helicase (DNaB).

39. 39 Figure 7.15

40. 40 Elongation of Replication After initiation, each replication fork contains two strands: - A leading strand, which is replicated continuously in the 5´-to-3´ direction - A lagging strand, which is replicated discontinuously in stages, each producing an Okazaki fragment - These are progressively stitched together to make a continuous unbroken strand.

41. 41 The cell coordinates the activity of two DNA pol III enzymes in one complex. - These two enzymes, together with DNA primase and helicase, form the replisome. The replisome ensures that the leading and lagging strands are synthesized simultaneously in the 5´-to-3´ direction. Replisome

42. 42 Figure 7.18a Figure 7.18b

43. 43 Elongation of Replication To remove RNA primers, cells use RNase H. A DNA pol I enzyme then synthesizes a DNA patch using the 3´ OH end of the preexisting DNA fragment as a priming site. Finally, DNA ligase repairs the phosphodiester nick using energy from NAD (in bacteria) or ATP (in eukaryotes).

44. 44 Figure 7.19

45. 45 Termination of Replication There are as many as 10 terminator sequences (ter) on the E. coli chromosome. A protein called Tus (terminus utilization substance) binds to these sequences and acts as a counter-helicase. Ringed catenanes formed at the completion of replication are separated by topoisomerase IV and the proteins XerC and XerD.

46. 46 Plasmids are extragenomic DNA molecules. - Much smaller than the chromosome - Usually circular - Need host proteins to replicate Plasmids Figure 7.23

47. 47 Plasmids Plasmids can replicate in two different ways: - Bidirectional replication - Starts at a single origin and occurs in two directions simultaneously - Rolling-circle replication: - Starts at a single origin and moves in only one direction

48. 48 Figure 7.24

49. 49 Plasmids have tricks to ensure their inheritance: - Low-copy-number plasmids segregate equally to daughter cells. - High-copy-number plasmids segregate randomly to daughter cells. Plasmids are advantageous under certain conditions: - Resistance to antibiotics and toxic metals - Pathogenesis - Symbiosis Plasmids can also be transferred between cells. Plasmids

50. 50 Restriction endonucleases cleave DNA at specific recognition sites, which are usually 4 to 6 bp and palindromes. - May generate blunt or staggered ends Figure 7.27A Analysis of DNA

51. 51 Agarose gel electrophoresis can be used to analyze the DNA fragments obtained by treatment with restriction enzymes. Figure 7.27B Analysis of DNA

52. 52 The polymerase chain reaction (PCR) can produce over a million-fold amplification of target DNA within a few hours. Figure 7.29 Analysis of DNA