Mechanisms of hexameric helicases

Abstract

Ring-shaped hexameric helicases are essential motor proteins that separate duplex nucleic acid strands for DNA replication, recombination, and transcriptional regulation. Two evolutionarily distinct lineages of these enzymes, predicated on RecA and AAAþ ATPase folds, have been identified and characterized to date. Hexameric helicases couple NTP hydrolysis with conformational changes that move nucleic acid substrates through a central pore in the enzyme. How hexameric helicases productively engage client DNA or RNA segments and use successive rounds of NTPase activity to power translocation and unwinding have been longstanding questions in the field. Recent structural and biophysical findings are beginning to reveal commonalities in NTP hydrolysis and substrate translocation by diverse hexameric helicase families. Here, we review these molecular mechanisms and highlight aspects of their function that are yet to be understood.

A fijivirus major viroplasm protein shows RNA-stimulated ATPase activity by adopting pentameric and hexameric assemblies of dimers

“…In contrast to the RNA-stimulated ATPase activity observed in P9-1, NSP2 has both NTPase and RNA triphosphatase (RTPase) activities, and RNA competes with NTPs for the active site (27) suggesting they may have different modes of action. Overall, P9-1 ATPase activity, RNA binding, in addition to its quaternary structures resembling the ring-like shape of hexameric helicases (45), support a key role for this protein in MRCV genome replication, reassortment, and packaging.…”

“…The P9-1ΔC-arm secondary structure elements comprise nine α-helices and nine β-strands organized as follows: αI (residues 51-61), αII (64)(65)(66)(67)(68), αIII (80-101), αIV (173-201), αV (205-209), αVI (211-228), αVII (238-247), αVIII (251-260), αIX (270-277), βA (14)(15)(16)(17), βB (45)(46)(47)(48), βC (114-117), βD (122-125), βE (156-159), βF (279-286), βG (289-297), βH (303-306), and βI (308-311) (Fig. 3A).…”

CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism

“…In the staircase model of hexameric helicase action, the ATPase motor domains of each subunit form a spiral around the DNA. ATP hydrolysis causes the ATPase domain at the bottom of the staircase to dissociate from the DNA and move to the top of the staircase ( 5 , 11 – 13 ). Exchange of adenosine diphosphate (ADP)/Pi for ATP enables the newly positioned “top subunit” to rebind DNA.…”

“…The SF1 and SF2 helicases are monomeric while the SF3 to 6 helicases are hexameric. The SF1 and SF2 helicases work by a “two-step” inchworm mechanism ( 8 , 9 ), while homohexameric helicases are proposed to function by a staircasing process wherein the hexamer forms a spiral of six subunits encircling the DNA, with sequential hydrolysis of ATP that leads to a hand-over-hand movement of subunits along ssDNA ( 5 , 10 – 13 ). Unlike homohexameric helicases, the six (Mcm2 to 7) motor subunits of CMG are each encoded by separate genes having distinct ATP sites.…”

“…In the cell, replicative hexameric helicases typically require a “loading” factor(s) that helps to place the helicase ring around DNA ( 5 , 12 ). In vitro, hexameric helicases can self-load onto ssDNA, but are typically very slow to bind DNA and therefore in vitro experiments often use a preincubation step with ATPγS or adenylyl-imidodiphosphate (AMP-PNP) to preload the helicase onto DNA before initiating unwinding with ATP.…”