Ch. 13 DNA Replication and Repair

Ch. 13 DNA Replication and Repair

1. DNA Replication

   • takes place by separation of the strands of the double helix, and synthesis of two daughter strands complementary to the two parental templates. This is known as semiconservative replication. 
   • Replication in Bacterial Cells
     • Replication forks and Bidirectional replication a. starts at origin site b. replication proceeds bidirectionally c. the replication forks are the areas where a pair of replicated segments come together to join the non replicated segments
     • Properties of DNA polymerases
       • a. it is DNA dependent because it uses DNA as a template b. dna polymerase requires a primer which provides the 3' hydroxyl terminus in which to add new nucleotides and it only occurs in 5' to 3' direction. 3 classes of polymerases in bacteria and none can intimate DNA chains. 
       • Structure and Function of DNA Polymerase
         • DNA polymerase 3
           • is the primary replication enzyme a. contains ten subunits that have various functions in replication b. by forming sliding clamp, Beta subunit maintains association between both the polymerase and the DNA template
         • DNA polymerase 1
           • Is involved in DNA repair and also removes RNA primers and replaces them with DNA
         • Exonuclease activities
           • Degrades nucleic acids by 5'to 3' nucleotides one at a time. DNA polymerase 1 has exonuclease activity in both directions. Each is assigned to a different domain of the enzyme. The 5'to 3' exonuclease of polymerase 1 degrades the primer.
     • Semidiscontinuous Replication
       • This refers to the lagging strands inability to continuously synthesize due to the polymerases inability to add nucleotides in the 3' to 5' direction. The lagging strand is made up into okazaki fragments and then sealed by DNA ligase. The RNA primers are then removed at the end of this process. 
     • Machinery operating at the replication fork
       • helicase, binding proteins, replisome, helicase and primase form primosome which moves across the lagging strand template. 
   • Replication in Eukaryotic cells
     •     Not as well understood as in prokaryotes. In vitro systems can be reconstituted with eukaryotic cellular extracts and/or purified components.     
     • Initiation of Replication in Eukaryotic Cells
       •     Not as well understood as in prokaryotes. In vitro systems can be reconstituted with eukaryotic cellular extracts and/or purified components. Eukaryote replicate genome in small portions termed replicons. Replicons located close together tend to be replicated simultaneously. Tightly compacted regions are replicated later in the cell cycle. Origins in yeast (ARSs). ARS prevents ORC from binding. Replication origins have not yet been identified in mammals.       
     • Restricting replication
       •       Origins pass through different states so that they only replicate their DNA once per cell cycle. Origin is bound by an ORC. Licensing factors bind to the origins. Activation factors bind to the chromosomes and induce the licensed origins to intiate replication.     
     • Chromatin Structure Relation to Replication
       •     DNA replication is associated with the opening of the nucleosome core particles. The assembly of DNA into nucleosomes is a very rapid event. Histones remain intact during replication and old and new histone octamers are distributed randomly between the two daughter duplexes.     

2. DNA Repair

   •     DNA repair is essential for cell survival. DNA is the cell molecule that is susceptible to environmental damage. Ionizing radiation, chemicals, thermal energy create various spontaneous lesions in DNA. Cells have a number of mechanisms to repair DNA and excision is the main one.     
   • Nucleotide Excision repair
     •     Removes bulky lesions such as pyrimidine dimers and chemically altered nucleotides. TFIIH links transcription and repair. An endonuclease makes nicks on two sides of the altered strand, after which damaged oligonucleotides are removed by helicase and the gap is filled by DNA polymerase and sealed with DNA ligase. Two pathways: preferential pathway selectively repairs the genes of greatest importance. Slower pathway repairs the rest of the genome.     
   • Base excision repair
     •     BER removes altered nucleotides that produce distortions of the double helix. DNA glycosylase recognizes the alteration and cleaves the base from the sugar. DNA glycosylases are specific for particular types of altered bases. Thymine occurs in DNA instead of uracil because otherwise the repair system would not have been able to distinguish between uracil and altered cytosine.     
   • Mismatch repair
     •       Corrects mistakes that escape the DNA polymerase proofreading. Repair enzymes recognize distortions caused by mismatched bases. The repair system must distinguish between parental and new strands. In bacteria, the two strands are distinguished by the presence of methyl groups on the parental strand. The mechanism of mismatch repair in eukaryotes involves the presence of certain replication proteins that remain bound to the daughter strands for a time.     

3. Between Replication and Repair