(15 points) Your graduate student discovers that this strain of E. coli has proteins with 100% sequence homology to polAex1 and polA+ but no protein homologous to any known DNA polymerase III: what is the likely role of polAex1 and polA+ in this strain. What effect would you expect this to have on DNA replication?
Added by Emily G.
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coli. polA+ corresponds to the normal Pol I, while polAex1 is an allele with altered exonuclease activity (typically a defective or modified exonuclease domain) but still retaining polymerase activity. Show more…
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In $E .$ coli, viable polA mutants have been isolated that produce a defective gene product with little or no $5^{\prime} \rightarrow 3^{\prime}$ polymerase activity, but normal $5^{\prime} \rightarrow 3^{\prime}$ exonuclease activity. However, no $p o l A$ mutant has been identified that is completely deficient in the $5^{\prime} \rightarrow 3^{\prime}$ exonuclease activity while retaining $5^{\prime} \rightarrow 3^{\prime}$ polymerase activity, of DNA polymerase I. How can these results be explained?
DNA polymerase I (Pol I) of E. coli consists of three functional parts (domains): 1. an N-terminal domain with 5' to 3' exonuclease activity required for removal of the RNA primer 2. a central domain responsible for 3' to 5' exonuclease proofreading 3. 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. Based on this given information, predict the ability of the following mutants to replicate DNA: a. a strain with a mutant gene encoding Pol I such that it no longer has polymerase activity (but retains both types of nuclease activities) b. a strain without RNaseH proteins c. a strain with a mutant gene encoding Pol I such that it no longer has 5' to 3' exonuclease activity (but retains 3' to 5' nuclease and polymerase activities) d. a strain with the mutant Pol I described in (3) and a strain lacking all RNaseH proteins. Explain your reasoning for each.
Josee P.
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 RNase H 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 RNase H 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 RNase H proteins.
Sri K.
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