In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we used cryo-EM to determine sub-4-Å -resolution structures of the E. coli DnaB,DnaC helicase,loader complex with nucleotide in pre-and post-DNA engagement states. In the absence of DNA, six DnaC protomers latch onto and crack open a DnaB hexamer using an extended N-terminal domain, stabilizing this conformation through nucleotide-dependent ATPase interactions. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to isomerize into a topologically closed, pre-translocation state competent to bind primase. Our data show how DnaC opens the DnaB ring and represses the helicase prior to DNA binding and how DnaC ATPase activity is reciprocally regulated by DnaB and DNA. Comparative analyses reveal how the helicase loading mechanism of DnaC parallels and diverges from homologous AAA+ systems involved in DNA replication and transposition.Supplemental Information includes seven figures and six videos and can be found with this article online at https://doi.

The adenine glycosylase MutY selectively initiates repair of OG:A lesions and, by comparison, avoids G:A mispairs. The ability to distinguish these closely related substrates relies on the Cterminal domain of MutY which structurally resembles MutT. To understand the mechanism for substrate specificity, we crystallized MutY in complex with DNA containing G across from the high-affinity azaribose transition state analog. Our structure shows that G is accommodated by the OG site and highlights the role of a serine residue in OG versus G discrimination. The functional significance of Ser308 and its neighboring residues was evaluated by mutational analysis, revealing the critical importance of a beta-loop in the C-terminal domain for mutation suppression in cells, and biochemical performance in vitro. This loop comprising residues Phe307, Ser308, and His309 (Geobacillus stearothermophilus sequence positions) is conserved in MutY but absent in MutT and other DNA repair enzymes, and may therefore serve as a MutY-specific target exploitable by chemical biological probes.Aberrant DNA modifications that arise from chemical reactions with exogenous and endogenous agents are considered "DNA damage" since these modifications put biological systems at risk. DNA repair enzymes mitigate this risk by counteracting chemical damage that otherwise would erode information content of DNA. 1 Guanine is particularly prone to oxidative damage due to its low redox potential. 2 Oxidation of G results in 8-oxo-7,8dihydroguanine (OG) which differs from G by only two atoms (Figure 1). The OG lesion is especially problematic because the syn conformer mispairs with adenine during DNA replication. The guanine oxidation (GO) repair pathway prevents mutations that otherwise would arise from OG template ambiguity (Figure 2). The GO repair pathway features ‡

In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of E. coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We identify a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as polymerase clamp loading and certain classes of DNA transposases.