Figure 4.

Purified recombinant hSKI binds preferentially ssDNA and telomeric DNA-RNA hybrids containing 3′ overhangs in vitro

(A) hSKI constructs. MBP, Maltose binding protein; His, 6x histidine; Flag, 3x flag.

(B) Coomassie blue SDS-PAGE gel showing purified hSKI (lane 10) and different purification fractions. PP, PreScission protease.

(C) Electrophoretic mobility shift assays showing binding of hSKI to different RNA and DNA substrates. Blue lines denote RNA, black lines denote DNA and asterisk indicates radioactive ³²P label. hSKI protein amounts are as indicated (nM) (D) Quantification of (C) showing % of nucleic acid binding calculated as protein bound substrate signal relative to free substrate signal (mean ± SEM, n = 2–4).

(E) Electrophoretic mobility shift assays showing binding of hSKI complex to different 3′ and 5′ overhang RNA/DNA substrates. Blue lines denote RNA, black lines denote DNA and asterisk indicates radioactive ³²P label.

(F) Quantification of (E) showing % of nucleic acid binding calculated as protein bound substrate signal relative to free substrate signal (mean ± SEM, n = 2). See also Figure S4.