RAPADILINO RECQL4 mutant protein lacks helicase and ATPase activity

Abstract

The RecQ family of helicases has been shown to play an important role in maintaining genomic stability. In humans, this family has five members and mutations in three of these helicases, BLM, WRN and RECQL4, are associated with disease. Alterations in RECQL4 are associated with three diseases, Rothmund-Thomson syndrome, Baller-Gerold syndrome, and RAPADILINO syndrome. One of the more common mutations found in RECQL4 is the RAPADILINO mutation, c.1390+2delT which is a splice-site mutation leading to an in-frame skipping of exon 7 resulting in 44 amino acids being deleted from the protein (p.Ala420-Ala463del). In order to characterize the RAPADILINO RECQL4 mutant protein, it was expressed in bacteria and purified using an established protocol. Strand annealing, helicase, and ATPase assays were conducted to characterize the protein's activities relative to WT RECQL4. Here we show that strand annealing activity in the absence of ATP is unchanged from that of WT RECQL4. However, the RAPADILINO protein variant lacks helicase and ssDNA-stimulated ATPase activity. These observations help explain the underlying molecular etiology of the disease and our findings provide insight into the genotype and phenotype association among RECQL4 syndromes.

Figure 1. Graphic representation WT and RAPA RECQL4

A, The intron-exon boundaries of RECQL4 gene. Exon 7 is deleted by the RAPA patient mutation, black filled rectangles. Exons 8–14, hatched, encode the RecQ helicase domain. B, Simply Blue stained SDS-PAGE gel of bacterially expressed and purified WT and RAPA RECQL4 proteins, 500 ng of each full length protein. C, Denaturation curves for 2× Sypro Orange control, dashed line, and WT RECQL4 plus 2× Sypro Orange, solid line. Insert shows original raw fluorescence values versus temperature, which the denaturation curve is extrapolated from.

Figure 2. RAPA RECQL4 possesses wild type levels of strand annealing

The strand annealing activities of the RECQL4 proteins were determined using radiolabeled T1 (0.5 nM) and cold B1 (1 nM), as described in the Material and Methods. A, a representative phosphorimage of the gel showing the relative single strand annealing activity of WT (lanes 2–5) and RAPA RECQL4 (lanes 6–9) in the absence of ATP. The position of the single stranded and forked dsDNAs are shown. B, graph of the quantification of three independent experiments of strand annealing in the absence of ATP. C, a representative phosphorimage of a gel showing the relative single strand annealing activity of WT (lanes 11–14) and RAPA RECQL4 (lanes 15–18) in the presence of ATP. D, graph of the quantification of three independent experiments of strand annealing in the presence of ATP. The mean and standard deviation are reported in B and D.

Figure 3. RAPA RECQL4 lacks helicase activity

The helicase activity of WT and RAPA RECQL4 was determined using the forked duplex B1/T1 (0.5 nM) as described in the Materials and Methods. A, a representative phosphorimage of a gel displaying the helicase activity of WT (lanes 2–6) and RAPA RECQL4 (lanes 7–11) is shown. The Δ symbol denotes the heat denatured DNA substrate. The position of the single stranded and forked dsDNAs are denoted by the images on the left side of the gel. B, a graph of the quantification of three independent helicase gels. The mean and standard deviation are reported.

Figure 4. Strand separating activity in the presence of excess single stranded DNA

The strand separating activity of WT and RAPA RECQL4 was determined using the labeled forked duplex B1/T1 (0.5 nM) in the presence of 12.5 nM cold B1 as described in the Materials and Methods. A, a representative phosphorimage of a gel displaying the strand separating activity of WT (lanes 2–3 and 9–10), RAPA RECQL4 (lanes 4–5 and 11–12), and K508M RECQL4 (lanes 6–7 and 13–14) in the absence and presence of ATP is shown. The Δ symbol denotes the heat denatured DNA substrate. The position of the single stranded and forked dsDNAs are denoted by the images on the left side of the gel. B and C, graph of the quantification of three independent gels each in the absence and presence of ATP, respectively. The mean and standard deviation are reported.

Figure 5. RAPA RECQL4 lacks ATPase activity

A, a representative phosphorimage of a TLC plate showing the relative ATPase activity of WT and RAPA RECQL4. For this assay a 61-mer ssDNA (8 pmol) was incubated with either WT or RAPA RECQL4 and 1 μCi [γ-³²P] ATP (PerkinElmer Life Sciences) in helicase assay buffer with 50 μM cold ATP for 1 h at 37 °C. The protein concentrations tested were 10, 50 and 100 nM. B, Graphic representation of the quantification of three independent ATPase experiments is shown. The mean and standard deviation are reported.

Figure 6. Comparison of RECQL4 activities in absence or presence of ssDNA

An image of RECQL4 protein is shown. The N-terminal and helicase domains are depicted as being responsible for the strand destabilization or helicase activity, respectively. The chart below image shows which activities are functional in the RECQL4 proteins, WT, K508M and RAPA, minus or plus ssDNA.