DNA polymerase I (Pol I) of E. coli consists of three functional parts (domains): an N-terminal domain with 5´ to 3´ exonuclease activities required for removal of the RNA primer, a central domain responsible for 3´ to 5´ exonuclease proofreading, and a C-terminal domain with polymerase activity. Pol I is thought to simultaneously remove RNA primers and fill in the gaps that result. A group of proteins known as RNaseH also have 5´ to 3´ exonuclease activity and can thus remove RNA primers. However, they lack the other two functions observed for Pol I. Predict the ability of the following mutants to replicate DNA: (1) a strain with a mutant gene encoding Pol I such that it no longer has polymerase activity (but retains both types of nuclease activities); (2) a strain without RNaseH proteins; (3) a strain with a mutant gene encoding Pol I such that it no longer has 5´ to 3´ exonuclease activities (but retains 3´ to 5´ nuclease and polymerase activities); (4) a strain with the mutant Pol I described in (3) and a strain lacking all RNaseH proteins.
DNA polymerase I (Pol I) of E. coli consists of three functional parts (domains): an N-terminal domain with 5´ to 3´ exonuclease activities required for removal of the RNA primer, a central domain responsible for 3´ to 5´ exonuclease proofreading, and a C-terminal domain with polymerase activity. Pol I is thought to simultaneously remove RNA primers and fill in the gaps that result. A group of proteins known as RNaseH also have 5´ to 3´ exonuclease activity and can thus remove RNA primers. However, they lack the other two functions observed for Pol I. Predict the ability of the following mutants to replicate DNA:
(1) a strain with a mutant gene encoding Pol I such that it no longer has polymerase activity (but retains both types of nuclease activities);
(2) a strain without RNaseH proteins;
(3) a strain with a mutant gene encoding Pol I such that it no longer has 5´ to 3´ exonuclease activities (but retains 3´ to 5´ nuclease and polymerase activities);
(4) a strain with the mutant Pol I described in (3) and a strain lacking all RNaseH proteins.
Introduction:
Synthesis of DNA requires the RNA primers to start the addition of deoxyribonucleotides. The deoxyribonucleotides are added in the direction of 3' to 5'. However, the template strand is read in the direction of 5' to 3' by the DNA polymerase. This orientation is in regards to the 5' phosphate group and the 3' hydroxyl group present on the ribose sugar of every nucleotide. DNA polymerase causes the addition of nucleotides to a 3' hydroxyl group. At the time of DNA synthesis, DNA polymerase reads the template strand and inputs the complementary deoxyribonucleotide. DNA polymerase does make mistakes while replicating DNA and often serves the proofreading activity. This permits the polymerase to recognize that the wrong base pair has been added and removes it by damaging the phosphodiester bond that was made. This is called exonuclease activity. After the DNA synthesis, the RNA primers must be removed and replaced by DNA.
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