DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain

Abstract

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA.

Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation

“…Indeed, the absence of neither PolQ nor PolN in several eukaryotes may suggest the functions provided by these Pols have been discarded several times during evolution. In all cases, how retention or discarding of PolQ and PolN relates to the presence or absence of a separate HelQ protein deserves further consideration, particularly since HelQ-like, but not PolQ- or PolN-like, proteins have been described in archaea ( 62 , 63 ), and C. elegans and D. melanogaster encode PolQ and HelQ-like proteins but not PolN. In this context, the distinct grouping of TbPolIE as one apparent paralogue of five kinetoplastid genes encoding only a Pol domain, and the presence of an isolated HelQ-like protein in all kinetoplastids, muddies any clarity in understanding the evolution of these seemingly connected proteins.…”

Determining the effects of DNA sequence on Hel308 helicase translocation along single-stranded DNA using nanopore tweezers

“…Hel308 helicase has five major domains (23): the two RecA folds which are present in SF1 and SF2 helicases and generate directed motion by an inch-worm mechanism (3,24), a winged-helix domain which promotes binding to duplex DNA (25), a ratchet domain that interacts with DNA bases via hydrogen bonding and stacking interactions at multiple sites with the DNA, and a helix-loop-helix domain that is autoinhibitory to unwinding activity (26). The crystal structure of Hel308 from Archaeoglobus fulgidus conjugated with DNA shows that Hel308 interacts directly with the DNA bases at ∼10 different sites (23).…”

Mechanistic insights into Lhr helicase function in DNA repair

“…Biochemical analysis of the Lhr-Core from the bacteria Mycobacterium smegmatis and Pseudomonas putida identified ATP-dependent ssDNA translocation with 3′ to 5′ directionality [ 1 , 9 , 10 ]. A crystal structure of bacterial Lhr-Core highlights significant similarities with the archaeal DNA repair helicase Hel308 [ 9 , 11 ], most notably in the orientation and interaction of its winged helix domain (WHD) with RecA-like domains typical of Ski2-like helicases [ 12 , 13 ].…”