Question

Following is a diagram of the general structure of the bacteriophage \(\lambda\) chromosome. Speculate on the mechanism by which it forms a closed ring upon infection of the host cell. \(5^{\prime}\) GGGCGGCGACCT-double-stranded region-3' 3'- double-stranded region-CCCGCCGCTGGA 5'

Short answer

Short Answer: Bacteriophage λ forms a closed ring upon infection of a host cell by the process of annealing its cohesive or "sticky" ends, which are complementary single-stranded overhangs at both ends of its linear chromosome. This process is driven by base pairing between the complementary overhangs, and the resulting nicks in the DNA backbone can be repaired by host cell enzymes like DNA ligase to complete the circularization of the chromosome.

Step-by-step solution

Overview of bacteriophage λ

Bacteriophage λ is a virus that infects the bacterium Escherichia coli (E. coli). When it comes to its chromosome structure, bacteriophage λ has a linear, double-stranded DNA molecule as its genetic material. However, upon infection of the host cell, this linear DNA undergoes a process that results in a circular form. In the exercise, we are provided with a simple representation of the λ chromosome, where the DNA starts with "5' GGGCGGCGACCT-double-stranded region-3'" and ends with "3'- double-stranded region-CCCGCCGCTGGA 5'". The double-stranded regions correspond to the complementary strands of DNA that need to be connected to form a closed ring.

Speculating on the circularization mechanism

There are a few mechanisms by which bacteriophage λ DNA can circularize upon host cell infection. Some of these mechanisms involve proteins and enzymes from both the host bacteria and the bacteriophage itself. Here, we will discuss two possible mechanisms for the formation of a closed ring in the λ chromosome. 1. Cohesive ends: Bacteriophage λ can form a circle due to the presence of cohesive ends, also known as "sticky ends". These are short, single-stranded overhangs at both ends of the λ chromosome. These overhangs are complementary to each other, enabling them to bind together and form a closed ring. Upon infection of the host cell, λ DNA enters the cell as a linear molecule, and its cohesive ends pair up in a process called "annealing" to form a circular chromosome. This process is driven by base pairing between the complementary single-stranded overhangs generated from the 5’-end GGGCGGCGACCT and 3’-end CCCGCCGCTGGA. 2. DNA ligase: Another possible mechanism of chromosome circularization involves host cell enzymes called DNA ligases. These enzymes facilitate the formation of covalent phosphodiester bonds between the ends of DNA molecules. After the annealing of cohesive ends, there might be a small nick in the DNA backbone, which is then repaired by the action of DNA ligase. This results in a fully closed, circular λ chromosome. In summary, bacteriophage λ can form a closed ring upon host cell infection through mechanisms such as complementary cohesive ends and the action of host cell enzymes like DNA ligase.

Key concepts

Cohesive Ends

In the context of bacteriophage \(\lambda\) life cycle, 'cohesive ends' play a pivotal role during the infection process. These are also known as 'sticky ends', which are short, single-stranded overhangs at each end of the viral DNA strand. Imagine two strands of a zipper that are designed to mesh with each other perfectly; similarly, cohesive ends are designed to bind with their exact complementary sequence. When the bacteriophage injects its DNA into a host cell, the cohesive ends productively align and anneal, essentially zipping up to convert the linear DNA into its circular form. This circularization is crucial for the virus's life cycle as it allows the viral DNA to mimic the host's circular DNA and integrate into the host genome, which is necessary for replication.

This mechanism is highly efficient, but it is not enough to stabilize the newly-formed circular DNA on its own. The process is initiated through natural base pairing, utilizing hydrogen bonds to bring the cohesive ends together. However, for the circular DNA to be fully stable and functional, it needs to be covalently sealed, which is where DNA ligase comes into play.

DNA Ligase

DNA ligase is an indispensable enzyme in the realm of molecular biology, particularly when it comes to stitching DNA fragments together. In the process of bacteriophage \(\lambda\) chromosome circularization, after the cohesive ends anneal to form the circular shape, a gap or 'nick' remains in the sugar-phosphate backbone of the molecule where the ends meet. DNA ligase is like a molecular seamstress, adept at repairing nicks in the DNA. It catalyzes the formation of phosphodiester bonds between the 3'-hydroxyl end of one nucleotide and the 5'-phosphate end of another, sealing the cohesive ends permanently and ensuring the integrity of the circular DNA.

This action is not only essential for the successful infection by bacteriophage \(\lambda\) but is also a key step in DNA replication, repair, and recombination in all living organisms. The reliable function of DNA ligase ensures the genetic material's continuity and stability, which is paramount for the propagation of genetic information.

Annealing Process

The annealing process is a phenomenon that serves as a foundation for the circularization of bacteriophage \(\lambda\)'s chromosome. Annealing refers to the pairing of complementary single-stranded DNA regions to form a double-stranded structure. This occurs as the cohesive ends of the bacteriophage \(\lambda\) come into proximity within the host cell. Due to the inherent properties of nucleotide bases, adenine (A) pairs only with thymine (T), and cytosine (C) pairs with guanine (G), which drives the accurate alignment and pairing of these sticky ends.

During viral infection, the single-stranded overhangs of the phage DNA undergo annealing, allowing the linear DNA to assume a circular form. The process is largely spontaneous, facilitated by the natural tendency of complementary bases to hydrogen-bond with each other. While enzymatic actions such as those by DNA ligase complete the circularization process, it is the initial annealing that sets the stage for the stable, covalently closed circle of viral DNA necessary for its integration and replication within the host.

Phosphodiester Bond Formation

Phosphodiester bond formation is the chemical backbone of DNA molecules. These covalent bonds link the 5'-phosphate group of one nucleotide to the 3'-hydroxyl group of the sugar in another. During the maturation of the bacteriophage \(\lambda\) chromosome, the annealed cohesive ends are transformed into a continuous double-stranded DNA molecule through the formation of phosphodiester bonds.

Phosphodiester bond formation is indispensable for the stabilization of the genetic material's structure, thereby allowing the bacteriophage to replicate within the host cell. The DNA ligase enzyme is the workhorse that catalyzes this bonding, ensuring that the integrity of the circular chromosome is maintained. DNA's stability, heredity function, and ability to store genetic information all hinge on the integrity of these bonds, showcasing the significance of phosphodiester bond formation in both viral replication and the broader spectrum of biological processes.