Genome Replication: Topological Problems

Primarily, three possible modes of replication were postulated:

Semi-conservative: According to the semi-conservative mode of replication, one of each parental strand is used as a template for the daughter strands during replication. It means that one of the strands will be completely new while the other will come from the parents.
Conservative: According to the conservative mode of replication, completely new daughter strands will be formed while the original parental strands remain conserved.
Dispersive: According to the dispersive mode of replication, after one round of replication, the daughter strands will have partly old DNA and partly new DNA forming hybrids.

Watson and Crick’s scheme for DNA replication: They proposed the double helical structure for the DNA. Along with this, they also proposed that Adenine always pairs with Thymine, Cytosine always pairs with Guanine, and vice-versa¹.

Schematic Diagram of Replication Fork showcasing different proteins and enzymes involved during replication initiation and elongation.

Topological problem and its solution

The most obvious problem during DNA replication is the possibility of daughter molecules getting entangled. Even more daunting the rotation that would occur for every 10 bp of the double helix. For instance, human chromosome 1 is approx. 250 Mb in length, meaning it would require 25 million rotations during its complete replication. Although hard to imagine in the limited constraints of nucleus present inside an already microscopic cell, it is not physically impossible for a linear DNA to unwind via rotation. In comparison, this rotation would be impossible for a circular double-stranded DNA molecule present in bacteria or viral genome that has no free ends.

Failed Solutions

Briefly, it was suggested that the problem with regard to the unwinding of the DNA could be solved if the DNA helix was paranemic (i.e. the two strands were lying in a side-by-side ribbon-like structure rather than in helix) rather than plectonemic (helical structure). However, this was rejected as it did not match with any of the experimental data pertaining to the DNA structure as well as the X-ray diffraction results.

Real Solution to Topology Problem

The correct solution came with the discovery of DNA topoisomerases which worked by breaking off one of the strands, passing the other strand through the gap and rejoining the first strand (breakage-and-reunion model). This reduced the torsional stress arising due to the rotation of the helix during unwinding.

DNA topoisomerases and its types

Topoisomerases are enzymes that work in the regulation of underwinding or overwinding of DNA. In order to solve the topological problems arising due to helix, topoisomerases bind to either single or double-stranded DNA and cut the phosphate backbone of the DNA. This breakage allows for the reduction of the torsional stress and releases the pressure arising due to unwinding

In order to prevent and correct these types of topological problems caused by the double helix, topoisomerases bind to either single-stranded or double-stranded DNA and cut the phosphate backbone of the DNA. In a nutshell, as their name suggests, they change the topology of the DNA without changing the chemical composition or connectivity of the DNA.

Types of topoisomerases:

They are divided into two types depending on the number of strands cut in one round of action.
Both of the enzymes utilize a conserved tyrosine, however, they are structurally and mechanistically different.

Type I:

Cuts one strand of the DNA, reduces the stress due to too much or too little twist, and then rejoins the cut strand.
Subdivided into two subclasses: Type IA and Type IB topoisomerase.

Type II:

Cuts both strand of the DNA, moves around another unbroken helix, and then rejoins the broken strands.
Also divided into two subclasses: Type IIA and IIB.

Topoisomerase	IA	IB	IIA	IIB
Metal Dependence	Yes	No	Yes	Yes
ATP Dependence	No	No	Yes	Yes
Single- or Double-Stranded cleavage?	SS	SS	DS	DS
Cleavage Polarity	5′	3′	5′	5′
Change in Linking Number	±1	±N	±2	±2