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Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA-damaging agent is present., Competing Interests: The authors have declared that no competing interests exist.

Initiation of bacteriophage Mu DNA replication by transposition requires the disassembly of the transpososome that catalyses strand exchange and the assembly of a replisome promoted by PriA, PriB, PriC and DnaT proteins, which function in the host to restart stalled replication forks. Once the molecular chaperone ClpX weakens the very tight binding of the transpososome to the Mu ends, host disassembly factors (MRFalpha-DF) promote the dissociation of the transpososome from the DNA template and the assembly of a new nucleoprotein complex. Prereplisome factors (MRFalpha-PR) further alter the complex, allowing PriA binding and loading of major replicative helicase DnaB onto the template promoted by the restart proteins. MRFalpha-PR is essential for DnaB loading by restart proteins even on the deproteinized Mu fork whereas MRFalpha-DF is not required on the deproteinized template. When the transition from transpososome to replisome was reconstituted using MRFalpha-DF and MRFalpha-PR, initiation of Mu DNA replication was strictly dependent upon added PriC and PriA helicase. In contrast, initiation on the deproteinized template was predominantly dependent upon PriB and did not require PriA's helicase activity. The results indicate that transition mechanisms beginning with the transpososome disassembly can determine the pathway of replisome assembly by restart proteins.

Bacteriophage Mu DNA synthesis is initiated during transposition by replication restart proteins PriA, DnaT and either PriB or PriC. The PriA-PriC pathway requires PriA's helicase activity and other host factors that promote the orderly transition from transpososome to replisome on the Mu DNA template. The host factor MRFα-PR, which removes obstacles to PriA binding and promotes the PriA-PriC pathway, was identified to be the translation initiation factor IF2. Purified isoform IF2-2, which is truncated at the N-terminal end, had full MRFα-PR activity whereas full-length IF2-1 was inactive. IF2-2 was bound to the Mu DNA template specifically at the step for prereplisome assembly. Prior steps in the orderly transition from transpososome were essential to promote efficient IF2-2 binding. Moreover, PriA helicase activity was subsequently needed to displace IF2-2, remodelling the template to permit replisome assembly. IF2's role in the transition mechanism as well as its function as G protein and translation factor suggest its potential to regulate DNA synthesis by this pathway. [ABSTRACT FROM AUTHOR]

Explores the recombinase transposition to replisome. Role of PriA helicase in DNA binding; Formation of liposome by the assembly of DNA polymerase III holoenzyme; Transition of DNA replication to DNA synthesis; Function of the transposome in Mu DNA replication.

Examines the initiation of DNA synthesis on forked DNA templates in the replication and maintenance of cellular chromosomes. Function of bacterial PriA protein; Promotion of duplex opening fro replisome assembly; Sites of homologous strand exchange.