Journal: Nucleic Acids Research
Article Title: Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands
Figure Lengend Snippet: Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
Article Snippet: ATPase assays Hydrolysis of ATP by Hel308a was measured spectroscopically using malachite green assays ( ). φX174 circular ssDNA and RFII circular double-stranded DNA (dsDNA) substrates were from New England Biolabs.
Techniques: Sequencing, SDS Page, Purification, Recombinant, Marker, Activity Assay, Derivative Assay