DNA Polymerase and Replication Flashcards
What was the main goal of Kornberg’s experiments on E. coli, and how did they approach it?
The goal was to isolate and identify DNA polymerase in E. coli. They grew E. coli, broke open the cells, prepared a soluble extract, and fractionated it repeatedly to isolate proteins, testing for DNA polymerase activity by looking for incorporation of radioactive nucleotides into DNA.
What was the purpose of fractionating the extract in Kornberg’s experiments?
Fractionation was used to isolate DNA polymerase based on its unique biophysical properties such as size, solubility, and binding specificity, ultimately creating a homogenous mixture containing the polymerase.
How did Kornberg’s team initially detect DNA polymerase activity in their assays?
They used radio-labeled nucleotides (thymidine or dTTP), mixed them with isolated fractions, and tested for incorporation of the nucleotides into polymerized DNA by checking for acid-insoluble DNA that could be further verified by DNase treatment.
What was the key improvement made in the second iteration of Kornberg’s experiment compared to the first?
The second iteration used P42-labeled dTTP (with a radioactive phosphate), which provided a direct substrate for DNA polymerase and produced a stronger decay signal for easier detection.
How did Kornberg verify that the acid-insoluble pellet contained polymerized DNA?
They treated the acid-insoluble pellet with DNase, which breaks down DNA into soluble nucleotides. If the pellet contained real polymerized DNA, the radioactive signal would shift from the pellet to the soluble fraction after DNase treatment.
What is the significance of Paula DeLucia and John Cairns’ discovery regarding DNA polymerase I?
They isolated a mutant strain of E. coli that lacked DNA polymerase I activity, showing that DNA polymerase I is not essential for replication of the chromosome, leading to the discovery of DNA polymerase III as the main enzyme responsible for DNA replication.
What role does DNA polymerase I play in E. coli if it is not the primary enzyme for DNA replication?
DNA polymerase I is primarily involved in cleanup processes during replication, recombination, and repair. It has 5’→3’ exonuclease activity for removing RNA primers and filling in gaps during replication.
What unique property does DNA polymerase I have that allows it to perform “nick translation”?
DNA polymerase I has 5’→3’ exonuclease activity, allowing it to remove nucleotides ahead of the growing DNA chain while synthesizing new DNA to fill in the gaps, particularly during the removal of RNA primers.
What is the significance of the Klenow fragment in DNA polymerase I?
The Klenow fragment is a large portion of DNA polymerase I that retains the 3’→5’ exonuclease proofreading activity and polymerase activity but lacks the 5’→3’ exonuclease domain responsible for nick translation.
What is the main function of DNA polymerase III in E. coli?
DNA polymerase III is the principal enzyme responsible for rapid and accurate replication of the E. coli chromosome, with high processivity and fidelity during DNA synthesis.
What are the roles of DNA polymerases IV and V in E. coli?
DNA polymerases IV and V are specialized polymerases involved in DNA repair, particularly in translesion synthesis, which helps bypass DNA damage caused by factors like UV radiation.
How did Kornberg’s fractionation experiments contribute to the discovery of DNA polymerase I?
Kornberg’s fractionation experiments systematically isolated proteins from E. coli, confirming the presence of DNA polymerase I by demonstrating that fractions could incorporate nucleotides into DNA in a polymerization assay.
Why is DNA polymerase I considered the prototype for all DNA polymerases?
DNA polymerase I was the first polymerase to be discovered, and it laid the groundwork for understanding the enzymatic and structural properties of DNA polymerases across different species.
How does the 5′→3′ exonuclease activity of DNA polymerase I differ from its 3′→5′ proofreading activity?
The 5′→3′ exonuclease activity removes nucleotides ahead of the polymerization site for nick translation, while the 3′→5′ exonuclease activity corrects errors during DNA synthesis by removing mismatched nucleotides.
What did Kornberg’s experiments reveal about the solubility differences between polymerized DNA and free nucleotides?
Polymerized DNA is acid-insoluble, while free nucleotides are acid-soluble. This difference allowed Kornberg to determine whether DNA polymerase activity was present by checking for radioactivity in the acid-insoluble fraction.
