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Chapter 20 DNA Metabolism Gene: A segment of DNA or RNA that encodes a functional biological product = Protein or RNA E. coli gene products are sometimes named after the gene: recA RecA Chromosomes: DNA macromolecules are folded into chromosomes. http://www.copernicusproject.ucr.edu/ssi/HighSchoolBioResources/DNA/chromosome.gif Genome: Sum of all genes and intergenic DNA. The Human Genome contains about 23,000 genes encoding proteins and about 10,000 genes encoding RNA. The human genome contains about 3 billion base pairs. Only about 2% of the genome encodes genes. The E. coli genome contains about 4.6 million base pairs and encodes 4403 genes. Figure 20.16 shows a Map of the E. coli circular, double-stranded chromosome and the positions of some genes important in DNA replication. Exonucleases: Enzymes that degrade DNA 5'3' or 3'5' from one end. Endonucleases: Enzymes that cleave interior phosphodiester bonds. DNA Polymerases are enzymes that synthesize DNA. Eukaryotes contain 5 DNA Polymerases. E. coli has 3 DNA polymerases. I II III Subunits 1 4 10 Rate Nucl/s 20 7 1000 Processivity 3-200 10,000 500,000 Processivity = the number of nucleotides added before the enzyme dissociates. DNA Polymerase I also has a 5'3' exonuclease activity needed in DNA replication, recombination, and repair. For e.g. DNA Pol cannot seal nicks in DNA strands. Replisome: A complex of proteins involved in DNA replication. It consists of: Helicases: Use ATP to dissociate DNA strands. Topoisomerases: Relieve topological stress due to strand separation. DNA-binding proteins: Keep the strands separated. Primases: Make short RNA primers. DNA Ligases: Seal breaks in the DNA. DNA Replication: The process by which a cell copies its DNA to make a new cell. Recall that DNA is a double helix of two, antiparallel complementary strands in which A H-bonds with T, and G H-bonds with C. Recall also that DNA replication is semiconservative i.e. each strand serves as a template for the synthesis of a new strand; half of the old DNA is passed on to the daughter cell and half remains with the parent cell. There are 3 stages in DNA replication: 1. Initiation: In order to make a copy of the DNA, the double stranded DNA is separated at a replication fork. Figure 20.3 The electron micrographs show replication forks where DNA is unwound and replicating. Replication is usually bidirectional for circular DNA Bi-directional Synthesis Replication Forks ie there are two replication forks. Replication has a definite starting point: Origin. In E. coli it is called oriC. About 20 DnaA-ATP proteins bind to the Origin of replication. The DnaA-complex denatures the Origin forming an Open Complex. Then DnaB aids the binding of DnaC (a helicase) which unwinds the DNA bidirectionally. Topoisomerase II and single-strand DNA binding proteins cause 1000s of BP to be unwound. Initiation is the only point of regulation of replication but it is not well understood. 2. Elongation: Replication is always in the 5' 3' direction in both strands. The leading strand is made continuously. The lagging strand is made in short pieces called Okazaki fragments. The fragments are later ligated together. Leading Strand: DnaG (a primase) synthesizes a 10-60 nucleotide RNA primer. Next, DNA-Pol-III adds nucleotides continuously. 3' 5' 3' 5' Primase 3' 5' RNA 3' 5' Pol III 3' 5' 5' 3' DNA 5' 3' Lagging Strand: A Primosome includes DnaB, DnaC, DnaG + 4 others. The Primosome moves with the replication fork in the 5'3' direction. A primase synthesizes an RNA primer and DNA Pol-III adds to it. 3' Primase 5' 3' 5' Pol III 3' 5' 5' 3' 3' 5' DNA Pol-I removes the RNA primer, 5'3', and replaces the RNA with DNA. 5' 3' Pol I 3' 5' 5' 3' Ligase 3' 5' 5' 3' 3' 5' DNA ligase seals the nick between 2 Okazaki fragments. DNA synthesis is carried out by enzymes called DNA polymerases. DNA polymerase DNAn + dNTP DNAn+1 + PPi ΔG'o = +2 kJ/mol To make a phosphodiester bond requires the input of +2 kJ/mol. The energy comes from pyrophosphate hydrolysis. PPi Pi + Pi ΔG'o = -30 kJ/mol So net the ΔG'o = -28 kJ/mol DNA Polymerase requires a template and a primer. i.e. it can only add to, not initiate, a new strand. Mechanistically, the 3'OH attacks the 5'-γPhosphate. 5 Error rates of polymerases: 1:10 Errors occur when bases tautomerize and incorrectly H-bond. Proofreading improves the error rates to 1:108. Errors are detected by the 3'5' Proofreading Exonuclease Active Site of DNA Pol. After the DNA has been replicated, DNA Pol-I can remove RNA or DNA paired to the template using its 5'3' exonuclease activity; then it extends the nontemplate DNA and moves the nick to where it dissociates. Fig. 20.13b "Nick Translation" DNA or RNA 5' Template DNA 3' Pol I new DNA 5' 3' When an error is detected the polymerase slides back, the 3' 5' exonuclease site removes the incorrect base, then polymerization continues. Further error correction improves the error rates to 1:1010 (see later). 3. Termination: Little is known about how the 2 circular DNAs are separated. DNA Repair: Mutations are permanent changes in the DNA sequence. There is a strong correlation between the accumulation of mutations in cells and cancer. Some changes cause the death of a cell. At least 4 different repair systems are known: Mismatch Repair , Base-Excision Repair: , Nucleotide-Excision Repair: , Direct Repair: