CHAPTER 6 DNA Structure Replication and Manipulation 6 1 Genome size can differ tremendously even among closely related organisms Viral genomes 100 1000 kb bacterial genomes 1 10 Mb eukaryotic genomes 10 100 Mb The C value paradox states that among eukaryotes there is no consistent relationship between the c value DNA content of haploid genome and the metabolic developmental or behavioral complexity of the organism In higher animals and plants actual number of genes has little relationship to genome size much of the DNA has functions other than coding for the amino acid sequence of proteins 6 2 DNA is a linear polymer of four deoxyribonucleotides Adenine Guanine are purines double ring Thymine Cytosine are pyrimidines single ring A nucleotide is a nucleoside plus a phosphate In a polynucleotide chain the phosphate attached to the 5 carbon of one sugar is linked to the hydroxyl group attached to the 3 carbon of the next sugar in line The chemical bonds by which the sugar components of adjacent nucleotides are linked through the phosphate groups are called phosphodiester bonds The terminal groups are a 5 phosphate and a 3 hydroxyl 6 3 Duplex DNA is a double helix in which the bases form hydrogen bonds There are ten bases per turn of the double helix Human DNA 39 G C Regions poor in G C content show dark bands when chromosomes are stained with Giemsa A T has two hydrogen bonds and G C has three hydrogen bonds 6 4 Replication uses each DNA strand as a template for a new one Nucleotides are added one at a time to the growing end of a DNA strand DNA Polymerase DNA replication is semi conservative the parental strands remain intact This was demonstrated by Meselson and Stahl using an ultracentrifuge N15 used for physical separation of DNA molecules instead of N14 equilibrium density gradient centrifugation DNA strands must unwind to be replicated The position along a molecule at which DNA replication begins is called a replication origin and the region in which parental strands are separating is called a replication fork The process of generating a new replication fork is initiation DNA nearly always replicates bidirectionally Rolling circle replication starts with a single strand cleavage at a sugar phosphate bond in a double stranded circle producing two chemically distinct ends a 3 end and a 5 end Eukaryotic DNA molecules contain multiple origins of replication Movement of each replication fork proceeds at a rate of approximately 10 to 100 nucleotide pairs per second 6 5 Many proteins participate in DNA replication Unwinding the double helix to separate the parental strands requires a helicase protein that hydrolyzes ATP to drive the unwinding reaction A single stranded DNA binding protein SSB stabilizes the DNA as single strands so they do not come together again spontaneously An enzyme called gyrase cleaves both strands of the DNA duplex swiveling the ends to relieve torsional stress and rejoining the strands Enzymes capable of catalyzing breaking and rejoining of DNA strands are known as topoisomerases Each new DNA strand or fragment is initiated by a short RNA primer DNA polymerase can only elongate an existing strand at the 3 end it cannot initiate synthesis of a new strand need RNA polymerase can initiate RNA synthesis without a primer DNA is initiated by a short stretch of primer DNA Primer synthesized by a multienzyme complex called a primosome 12 nucleotides of RNA DNA polymerase has a proofreading function that corrects errors in replication The enzyme DNA polymerase forms the sugar phosphate bond phosphodiester bond between adjacent nucleotides DNA synthesis proceeds by the elongation of primer chains Polymerase III is the major replication enzyme elongation of DNA molecules initiation of replication fork at origins of replication and addition of deoxynucleotides to the RNA primers The key enzyme responsible for the replication of chromosomal DNA is called polymerase delta Major DNA polymerases have an exonuclease activity that breaks phosphodiester bonds in the sugar phosphate backbones also the editing function or proofreading function The genetic significance of the proofreading function is that it is an error correcting mechanism that serves to reduce the frequency of mutation resulting from the incorporation of incorrect nucleotides in DNA replication One strand of replicating DNA is synthesized in pieces precursor fragments The leading strand is continuous and the lagging strand is discontinuous or Ozaki fragments Precursor fragments are joined together when they meet Final stitching requires removal of RNA primer replacement with a DNA sequence and joining where adjacent DNA fragments come into contact RPA Replication protein A joins the complex by unwinding the RNA and a short segment of DNA from the double helix and stabilizes the unwound single strand by binding to it RPA also recruits endonucleases that cleave the unwound single strand from the double helix The polymerase complex replaces the excised segment with DNA and DNA ligase catalyzes the formation of the final bond connecting the two precursor fragments 6 6 Knowledge of DNA structure makes possible the manipulation of DNA molecules Single strands of DNA and RNA with complementary sequences can hybridize The process of renaturing DNA strands from two different sources is called nucleic acid hybridization A probe is denatured DNA where each molecule has been labeled with radioactive atoms or light emitting molecules DNA fragments can hybridize only if the length of the region in which they can pair is long The lower the temperature and higher the salt concentration the greater the proportion of mismatches that are tolerated Restriction enzymes cleave duplex DNA at particular nucleotide sequences The nucleotide sequence recognized for cleavage by a restriction enzyme is called the restriction site of the enzyme Sticky ends are small single stranded overhangs that are complementary in base sequence to the other end left by asymmetrical restriction sites Enzymes that have symmetrical cleavage sites yield DNA fragments that have blunt ends Restriction enzymes recognize a single restriction site without regard to the source of the DNA and the number of cuts in the DNA is determined by the number of restriction sites present The DNA fragment produced by a pair of adjacent cuts in a DNA molecule is called a restriction fragment a particular restriction enzyme produces a unique set of fragments for a particular