Question

8) Which of the following reactions with covalently closed, circular double stranded DNA (ccc dsDNA) does NOT result in a new linking number? Select one choice from the list below (A-D) and explain your answer.

A. incubation with a type I topoisomerase

B. incubation with a type II topoisomerase

C. incubation with ethidium bromide

D. a brief incubation with DNase I followed by resealing the breaks in the phosphodiester backbone with another enzyme (DNA ligase)

Answer 1

A. Incubation with a type I topoisomerase:

Topo isomerase I generally change the Linking number.

Reason: TopoisomeraseI binds to the DNA duplex and introduces a break in one of the stand. Topoisomerase then tightly binds with the two broken ends. The intact opposite strand then passes through the break. The enzyme then rejoins the ends and ultimately there is a change in turn of the plasmid. Change in linking number -1

B.Incubation with a type II topoisomerase:

It binds to the positive node of a cross over point of and forms a double-stranded break. It then passes through the intact duplex through the transient break before resealing. The net effect changes in positive node to a negative node change in linking number -2

C.EtBr unwinds DNA as it intercalates between bases.DNA unwinding is nothing but negative supercoiling.The unwinding angle is about 28-30 degrees per molecule of EtBr, Around 12-14 molecules of EtBr will efficiently unwind DNA by one turn (Change of Lk = -1). Hence, in a covalently closed circular DNA the binding of EtBr (or unwinding of DNA) must be compensated for by equivalent positive supercoiling.