Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif

Abstract

The structure and integrity of DNA is of considerable biological and biomedical importance, and it is therefore critical to identify and to characterize enzymes that alter DNA structure. DNA helicases are ATP-driven motor proteins that unwind DNA. Conversely, HepA-related protein (HARP) protein (also known as SMARCAL1 and DNA-dependent ATPase A) is an annealing helicase that rewinds DNA in an ATP-dependent manner. To date, HARP is the only known annealing helicase. Here we report the identification of a second annealing helicase, which we term AH2, for annealing helicase 2. Like HARP, AH2 catalyzes the ATP-dependent rewinding of replication protein A (RPA)-bound complementary single-stranded DNA, but does not exhibit any detectable helicase activity. Unlike HARP, however, AH2 lacks a conserved RPA-binding domain and does not interact with RPA. In addition, AH2 contains an HNH motif, which is commonly found in bacteria and fungi and is often associated with nuclease activity. AH2 appears to be the only vertebrate protein with an HNH motif. Contrary to expectations, purified AH2 does not exhibit nuclease activity, but it remains possible that AH2 contains a latent nuclease that is activated under specific conditions. These structural and functional differences between AH2 and HARP suggest that different annealing helicases have distinct functions in the cell.

Fig. 1.

Purification of human AH2. (A) Schematic diagrams of human HARP and AH2. Unique features of HARP include the N-terminal RPA-binding motif as well as two conserved HARP domains, which are found in HARP proteins. AH2 has an HNH motif in its C-terminal region. The positions of the motifs in HARP and AH2 are indicated. (B) Comparison of the HNH motif in AH2 with those in the MnlI and colicin E9 (ColE9) nucleases. The sequences of the HNH motifs in AH2 from humans (Homo sapiens), mouse (Mus musculus), western clawed frog (Xenopus tropicalis), zebra finch (Taeniopygia guttata), and Asian rice (Oryza sativa) are shown. This figure shows that the key H-N-H residues are conserved among AH2 proteins. The HNH consensus is from ref. . (C) Polyacrylamide-SDS gel electrophoresis of purified FLAG-tagged human AH2.

Fig. 2.

AH2 protein binds selectively to fork DNA. (A) AH2 binds with higher affinity to fork DNA than to ssDNA or dsDNA. Gel mobility shift experiments were performed with 30-nt ssDNA, 30-bp dsDNA, or 30-nt fork DNA that is identical to the dsDNA except for a 9-nt mismatch at one end. The relative concentrations of AH2 are shown above the lanes; the actual concentrations of AH2 are 0, 0.05, 0.1, 0.2, and 0.4 nM. (B) The ATPase activity of AH2 is stimulated to a greater extent by fork DNA than by ssDNA or dsDNA. The DNA substrates used in the ATPase assays are identical to those used in A, except that the DNA samples were not radiolabeled. Error bars represent SD (N = 3).

Fig. 3.

AH2 is an ATP-dependent annealing helicase. (A) Schematic diagram of the annealing helicase assay. (B) AH2 catalyzes the rewinding of DNA in an ATP-dependent manner. Annealing helicase assays were carried out in the presence or absence of the indicated factors, and the resulting DNA species were resolved by agarose gel electrophoresis. An equimolar concentration of UTP was used as a control for the absence of ATP.

Fig. 4.

AH2 is neither an RPA-removing enzyme nor a conventional helicase. (A) AH2 does not mediate the displacement of RPA from DNA. Gel mobility shift experiments were performed with radiolabeled bubble DNA that contains two high-affinity sites for RPA (the two 32-nt ssDNA segments) and for AH2 (the two DNA forks). A 10-fold molar excess of unlabeled d(CT)₃₀ was added in the binding reactions subsequent to the binding of RPA to the probe DNA. AH2 (3 nM), RPA (3 nM), and ATP (1.5 mM) were included, as indicated. The apparent compositions of the shifted complexes are specified. (B) AH2 does not exhibit helicase activity. Helicase assays were performed with partial duplex DNA substrates, as indicated, containing a radiolabeled oligonucleotide annealed to single-stranded phiX174 DNA. Purified AH2 or RecQ (as a positive control for a helicase) were incubated with the DNA substrates in the absence or presence or ATP. The products were resolved by electrophoresis in a 10% polyacrylamide gel.

Fig. 5.

Examination of differences between AH2 and HARP. (A) Unlike HARP, AH2 does not associate with RPA. Purified FLAG-tagged versions of wild-type HARP, mutant HARP lacking the conserved N-terminal RPA-binding motif, and wild-type AH2 were incubated with purified recombinant RPA, and then immunoprecipitated with anti-RPA2 resin. After the beads were washed, the bound proteins were eluted and detected by Western blot analysis with either anti-RPA2 or anti-FLAG. (B) AH2 does not exhibit nuclease activity with E. coli genomic DNA. AH2 (150 nM) or KpnI (15 u), an HNH nuclease, was incubated with E. coli genomic DNA (500 ng) for 2 h, and the digested DNA was resolved by agarose gel electrophoresis. ATP (1.5 mM final concentration) was included in the reactions where indicated. The reaction medium contained 10 mM Mg(II) and, where indicated, the following divalent metal cations: Ca(II) (1 mM), Co(II) (20 μM), Ni(II) (20 μM), and Zn(II) (20 μM).