Answer to Question #250807 in Microbiology for Pri

Question #250807

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)

Expert's answer

As per some studies Escherichia coli polA1 mutant, which lacks polymerase activity but has 5′-3′ exonuclease activity, was able to grow, although it accumulated many Okazaki fragments, probably due to its inability to fill gaps. In addition, the polA mutant, which lacks 5′-3′ exonuclease activity at 43°C, also accumulated Okazaki fragments and could not grow at high temperatures.

RNase H cleaves the RNA strand of RNA/DNA hybrids and plays a role in removing RNA primers of Okazaki fragments, although it cannot process a few ribonucleotides from the DNA-RNA junction sites.

The isolation of a double mutant of polA and rnh (which encodes RNase H) made it possible to detect RNA primers and contributed to the determination of the RNA primer length as 9 to 12 nucleotides.

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