Ch. 13 DNA Replication

• Genome:

  • All DNA present in a cell or virus
  • Bacteria and Archaea generally have one set (haploid 1N)
  • Eukaryotes have two sets (Diploid - 2N)
• Genotype:

  • Specific set of genes an organism possesses
• Phenotype:

  • Collection of observable characteristics
• DNA as Genetic Material:

  • Griffith in 1928 observed the change of non-virulent organisms into virulent ones via “transformation”
  • MacLeod and McCarty in 1944 showed that the transforming principle was DNA
• DNA and RNA Structure:

  • DNA and RNA are polymers of nucleotides linked together by phosphodiester bonds
  • DNA and RNA differ in the nitrogenous bases they contain, the sugars they contain, and whether they are single or double-stranded
• DNA Structure:

  • Polymer of nucleotides with bases adenine, guanine, cytosine, and thymine
  • Sugar is deoxyribose
  • Phosphate is esterified to sugar carbon
  • Double-stranded helix with base pairing: adenine and thymine pair by 2 hydrogen bonds, guanine and cytosine pair by 3 hydrogen bonds
• RNA Structure:

  • Polymer of nucleotides containing the bases adenine, guanine, cytosine, and uracil
  • Sugar is ribose
  • Most RNA molecules are single-stranded; some are double-stranded
  • Three different types: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA)
• Protein Structure:

  • Polymers of amino acids linked by peptide bonds
  • Amino acids have a central carbon with carboxy group (C-terminal), amino group (N-terminal), and side chain
  • Amino acids can be polar, non-polar, or charged depending on the side chain
• DNA Replication:

  • Complex process involving numerous proteins to ensure accuracy
  • Synthesis is semi-conservative; each daughter cell obtains one old and one new strand
• Patterns of DNA Synthesis:

  • DNA in most Bacteria is circular with bidirectional replication from a single origin
  • DNA polymerase catalyzes synthesis of a complementary strand of DNA in the 5’ to 3’ direction forming phosphodiester bonds
• Replication Machinery - Polymerases:

  • DNA polymerase catalyzes synthesis of a complementary strand of DNA
  • DNA synthesis occurs in the 5’ to 3’ direction forming phosphodiester bonds
  • Enzymes require a template, a primer, and dNTPs (dATP, DTTP, DCTP, dGTP)
• Replication Machinery – The Polymerase Pieces:

  • E. coli has 5 DNA polymerases with polymerase III playing the major role in replication
  • DNA polymerase holoenzyme is a complex of 10 proteins with 3 proteins forming the core enzyme
• DNA Synthesis – Events at the Replication Fork:

  • DNA polymerase synthesis is in the 5’ to 3’ direction only
  • Lagging strand is synthesized in short fragments called Okazaki fragments
  • DNA ligase forms a phosphodiester bond between 3’-hydroxyl of growing strand and 5’-phosphate of an Okazaki fragment
• Termination of Replication in E. coli:

  • Replication stops when the replisome reaches the termination site (ter) on DNA
  • Catenanes form when the two circular daughter chromosomes do not separate
  • Topoisomerases temporarily break the DNA molecules so the strands can separate
• Replication of Linear Chromosomes:

  • Shortening of chromosomes after each round of replication
  • Solved in eukaryotes by telomerase enzyme
  • Solved in bacteria by disguising the ends of the linear chromosome
• Gene Structure:

  • The basic unit of genetic information
  • Linear sequence of nucleotides with a fixed start point and end point
  • Codons are found in mRNA and code for single amino acids
• Transcription:

  • RNA synthesis under the direction of DNA
  • Three types of RNA are produced: mRNA, tRNA, rRNA
  • mRNA carries the message for protein synthesis
  • tRNA carries amino acids during protein synthesis
  • rRNA molecules are components of ribosomes
• Transcription in Bacteria – RNA Polymerase:

  • Most bacterial RNA polymerases have a core enzyme composed of 5 chains and a sigma factor
  • The holoenzyme (core enzyme + sigma factor) is required to begin transcription
• Transcription Initiation:

  • Promoter is the site where RNA polymerase binds to initiate transcription
  • Promoter has a specific sequence before the transcription starting point and a Pribnow box
• Transcription Elongation:

  • RNA polymerase unwinds the DNA and produces a transcription bubble
  • Moves with the polymerase as it transcribes mRNA from the template strand
• Transcription Termination:

  • Occurs when core RNA polymerase dissociates from the template DNA
  • Some terminators require the aid of the rho factor for termination
• DNA structure:

  • Double-stranded helix
  • Base pairing: adenine pairs with thymine by 2 hydrogen bonds, guanine pairs with cytosine by 3 hydrogen bonds
  • Major and minor grooves form when the two strands twist around each other
• Other replisome proteins:

  • Helicases unwind DNA strands
  • Single-stranded binding proteins (SSB) keep strands apart for replication
  • Topoisomerases break one strand of DNA to prevent supercoiling
  • DNA gyrase introduces negative supercoiling to compact bacterial chromosome
  • Primase synthesizes short complementary strands of RNA primers (~10 nucleotides) needed by DNA polymerase
• Events at the replication fork in E. coli:

  • DnaA proteins bind oriC causing bending and separation of strands
  • DnaB and other helicases separate strands, SSB attach
  • Primase synthesizes RNA primer
  • Lagging and leading strands are synthesized
  • DNA polymerase I removes RNA primers, fills gaps with DNA
  • Okazaki fragments are joined by DNA ligase
• Linking the fragments:

  • DNA ligase forms a phosphodiester bond between 3’-hydroxyl of growing strand and 5’-phosphate of an Okazaki fragment
• Protein-coding genes:

  • Template strand of DNA directs RNA synthesis in the 3’ to 5’ direction
  • Complementary DNA strand is the coding strand, same nucleotide sequence as mRNA (except in thymine)
• Protein-coding genes - Starts and Stops:

  • Promoter is located at the start of the gene, the recognition/binding site for RNA polymerase
  • Leader sequence is transcribed into mRNA but not translated into amino acids
  • Begins with DNA sequence 3’-TAC-5’, produces codon AUG, codes for N-formylmethionine, and ends with a stop codon
• tRNA and rRNA genes:

  • DNA sequences that code for tRNA and rRNA are considered genes
  • Genes coding for tRNA may code for more than a single tRNA molecule or type of tRNA
  • Genes coding for rRNA are transcribed as single, large precursor
• Transcription in bacteria:

  • Polycistronic mRNA often found in bacteria and archaea
  • Contains directions for more than one polypeptide catalyzed by a single RNA polymerase
• RNA polymerase


  An enzyme that synthesizes RNA from a DNA template during transcription, reading the DNA template strand in the 3' to 5' direction and adding RNA nucleotides one by one to the 5' end of the growing RNA strand.
• RNA primers


  Short sequences of RNA nucleotides synthesized by primase during DNA replication to provide a starting point for DNA polymerase.
• DNA primase


  An enzyme that synthesizes RNA primers during DNA replication, providing a starting point for DNA polymerase.
• DNA ligase


  An enzyme that catalyzes the formation of a phosphodiester bond between two adjacent nucleotides on a DNA strand
• Phosphodiester bond


  A covalent bond that links two adjacent nucleotides together, forming a continuous DNA strand
• Lagging strand


  The DNA strand that is synthesized in the opposite direction of the leading strand during DNA replication
• Okazaki fragments


  Short, discontinuous stretches of DNA synthesized on the lagging strand during DNA replication
• RNA primer


  A short RNA sequence that primes the synthesis of a new DNA strand during DNA replication
• RNA primer


  A small RNA molecule that acts as a starting point for DNA synthesis during DNA replication