The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader.

In many bacteria and eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+(ATPases Associated with various cellular Activities) ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of Escherichia coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We have identified 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 that elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as in polymerase clamp loading and certain classes of DNA transposases.

Structural Basis for Finding OG Lesions and Avoiding Undamaged G by the DNA Glycosylase MutY.

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 C-terminal 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 analogue. 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 ß 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.

Physical Basis for the Loading of a Bacterial Replicative Helicase onto DNA.

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.

A genome-wide linkage study in families with major depression and co-morbid unexplained swelling.

Major depressive disorder (MDD) is a common heritable condition. The diversity of the phenotype coupled with aetiological and genetic heterogeneity present formidable obstacles in the search for causative genetic loci. Studies of large families with many affected individuals, and the selection of well-defined clinical subgroups of depression, are two ways to reduce this complexity. Unexplained swelling symptoms (USS) are common in women and many patients give a strong personal and family history of depression. Co-morbid depression and swelling symptoms define a useful sub-phenotype for investigating genetic factors in depression. We have completed a genome-wide linkage analysis using 371 microsatellite markers in four families where MDD is co-morbid with USS. Of 47 affected individuals, 28 had both MDD and unexplained swelling, 11 had symptoms of swelling alone, and 8 had MDD alone. Parametric marker-specific analysis identified one suggestive locus, D8S260 (LOD = 2.02) and non-parametric multipoint variance component analysis identified a region on 7p (LOD = 2.10). A 47 cM suggestive linkage region on chromosome 14q (identified by both parametric and non-parametric methods) was identified and investigated further with fine-mapping markers but the evidence for linkage to this region decreased with increased marker information content.