DNA Replication II: Detailed Mechanism (CMB part 19)

Primer synthesis in E. coli involves the primosome, which consists of the DNA helicase DnaB and the primase DnaG. The assembly of the primosome at the origin of replication, oriC, proceeds as follows: DnaA binds to oriC at specific sites known as dnaA boxes and collaborates with RNA polymerase and HU protein to melt a DNA region adjacent to the leftmost dnaA box. Subsequently, DnaB associates with the open complex and promotes the binding of the primase to complete the primosome. The primosome remains attached to the replisome, repeatedly initiating Okazaki fragment synthesis on the lagging strand. Additionally, DnaB exhibits helicase activity, unwinding the DNA as the replisome advances.In the case of the SV40 origin of replication, it is located adjacent to the viral transcription control region. Replication initiation relies on the viral large T antigen, which binds within the 64-bp minimal ori at two adjacent sites. This antigen also possesses helicase activity, creating a replication bubble within the minimal ori. Priming is performed by a primase associated with the host DNA polymerase α.Yeast origins of replication are found within autonomously replicating sequences (ARSs), which consist of four key regions: A, B1, B2, and B3. Region A, a 15-bp sequence, contains an 11-bp consensus sequence that is highly conserved across ARSs. Region B3 may contribute to a critical DNA bend within ARS1.The pol III holoenzyme synthesizes DNA at a rate of approximately 730 nucleotides per second in vitro, slightly slower than the nearly 1000 nucleotides per second observed in vivo. This enzyme is highly processive both in vitro and in vivo. The pol III core (αε or αεθ) alone lacks processivity and can only replicate short DNA segments before dissociating from the template. However, when combined with the β-subunit, the core achieves processive replication at a rate approaching 1000 nucleotides per second. The β-subunit forms a dimer that takes on a ring-like structure, encircling the DNA.