HARP is an ATP-driven annealing helicase

Abstract

DNA-dependent adenosine triphosphatases (ATPases) participate in a broad range of biological processes including transcription, DNA repair, and chromatin dynamics. Mutations in the HepA-related protein (HARP) ATPase are responsible for Schimke immuno-osseous dysplasia (SIOD), but the function of the protein is unknown. We found that HARP is an ATP-dependent annealing helicase that rewinds single-stranded DNA bubbles that are stably bound by replication protein A. Other related ATPases, including the DNA translocase Rad54, did not exhibit annealing helicase activity. Analysis of mutant HARP proteins suggests that SIOD is caused by a deficiency in annealing helicase activity. Moreover, the pleiotropy of HARP mutations is consistent with the function of HARP as an annealing helicase that acts throughout the genome to oppose the action of DNA-unwinding activities in the nucleus.

Fig. 1

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

Fig. 2

HARP is an ATP-dependent annealing helicase. (A) HARP catalyzes the rewinding of DNA in an ATP-dependent manner. Annealing helicase assays, as depicted in fig. S3, 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. (B) HARP does not catalyze the ATP-dependent 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 single-stranded DNA segments) and for HARP (the two DNA forks). HARP (2 nM), RPA (3 nM), and ATP (1.5 mM) were included, as indicated. The apparent compositions of the shifted complexes are specified. Quantitation of the bands is shown in fig. S5. (C) Rad54, a member of the SNF2 family that translocates along DNA, does not exhibit annealing helicase activity. Annealing helicase assays were performed as in A with RPA and an equimolar concentration of Rad54 or HARP, where indicated.

Fig. 3

Mutant HARP proteins bind selectively to fork DNA, but exhibit less ATPase activity than wild-type HARP. The R764Q mutation results in a stronger SIOD phenotype than the R586W mutation (5). (A, B) The mutant HARP proteins bind with higher affinity to fork DNA than to single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). These gel mobility shift experiments were performed as in Fig. 1A. (C, D) The R586W HARP protein has low ATPase activity, whereas the R764Q HARP protein has no detectable ATPase activity. ATPase assays were carried out as in Fig. 1B. Error bars represent SD (N = 3).

Fig. 4

Mutant HARP proteins are defective in annealing helicase activity. The R586W HARP protein has less annealing helicase activity than wild-type HARP, whereas the R764Q HARP protein has no detectable annealing helicase activity. Annealing helicase assays were performed as in Fig. 2. All reactions contained plasmid DNA, RPA, and topoisomerase I. UTP was used as a control for the absence of ATP.