M13 Bacteriophage - Replication in E. Coli

Replication in E. Coli

Below are steps involved with replication of M13 in E. coli.

• Viral (+) strand DNA enters cytoplasm
• Complementary (-) strand is synthesized by bacterial enzymes
• DNA Gyrase, a type II topoisomerase, acts on double-stranded DNA and catalyzes formation of negative supercoils in double-stranded DNA
• Final product is parental replicative form (RF) DNA
• A phage protein, pII, nicks the (+) strand in the RF
• 3'-hydroxyl acts as a primer in the creation of new viral strand
• pII circulizes displaced viral (+) strand DNA
• Pool of progeny double-stranded RF molecules produced
• Negative strand of RF is template of transcription
• mRNAs are translated into the phage proteins

Phage proteins in the cytoplasm are pII, pX, and pV, and they are part of the replication process of DNA. The other phage proteins are synthesized and inserted into the cytoplasmic or outer membranes.

• pV dimers bind newly synthesized single-stranded DNA and prevent conversion to RF DNA
• RF DNA synthesis continues and amount of pV reaches critical concentration
• DNA replication switches to synthesis of single-stranded (+) viral DNA
• pV-DNA structures from about 800 nm long and 8 nm in diameter
• pV-DNA complex is substrate in phage assembly reaction