Question

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)

Answer 1

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.