Auto-inhibition of E. coli Rep Monomer Helicase Activity by its 2B Sub-domain

2 DNA helicases catalyze separation of double helical DNA into its complementary single strands, a process essential for DNA replication, recombination, and repair. The E. coli Rep protein, a superfamily 1 (SF1) DNA helicase, functions in DNA replication restart and is required for replication of several bacteriophages. Monomers of Rep do not display helicase activity in vitro; in fact, DNA unwinding requires Rep dimerization. Here we show that removal of the 2B sub-domain of Rep to form Rep∆2B activates monomer helicase activity, albeit with limited processivity. Although both full length Rep and Rep∆2B monomers can translocate with 3' to 5' directionality along ss-DNA, the 2B sub-domain inhibits the helicase activity of full length Rep. This suggests an auto-regulatory mechanism for Rep helicase, which may apply to other non-hexameric helicases, whereby helicase activity is regulated by the rotational conformational state of the 2B sub-domain; formation of a Rep dimer may relieve the auto-inhibition by altering the 2B sub-domain orientation. DNA helicases are a ubiquitous class of enzymes that utilize the binding and hydrolysis of nucleoside triphosphates to catalyze the separation of the DNA double helix into its complementary single strands. This process is essential for DNA replication, recombination, and repair (1-3) and defects in some human DNA helicases are linked to human diseases (4-6). DNA helicases are classified into superfamilies (SF) based on their primary structure, with the majority belonging to the SF1 and SF2 superfamilies (7). Some DNA helicases function as hexamers (8); others, such as the E. coli SF1 helicases Rep (9) and UvrD (10) and the SF2 hepatitis C viral (HCV) NS3 helicase (11) function as dimers in vitro, while others, such as the SF1 phage T4 Dda helicase (12) show limited 3 activity as monomers in vitro. The SF1 B. stearothermophilus PcrA helicase has been proposed to function as a monomer (13), although this has not been demonstrated experimentally. Possible roles for oligomerization in helicase activity have been discussed (3, 8, 14). The E. coli Rep protein (673 amino acids), a 3' to 5' SF1 DNA helicase (3, 14), is involved in replication restart (15) and is required for replication of some bacteriophages (16). Rep exists as a monomer in solution in the absence of DNA; however, in vitro, Rep monomers are inactive as helicases and Rep dimerization is required for processive DNA unwinding (9, 17). E. coli Rep is structurally homologous (18) to B. stearothemophilus unpublished …