Figure 1.
Rep68 oligomerizes in the presence of both sequence-specific and non-specific DNA.
Rep68 (16.6 µM: 1 mg/ml) was incubated in the absence (A) or presence of 2.8 µM ssDNA (B), 2.8 µM RBS dsDNA (C), or 2.8 µM non-specific dsDNA (D). Fifty µL of sample was chromatographed on a Superose 6 10/300 GL column with a flow rate of 0.5 mL/min. Protein elution was followed by UV detection at 280 nm. X axis represents the elution volume (Ve, in ml). Molecular weight standards and V0 position are shown in panel A. Sedimentation velocity data was obtained for the Rep68-RBS (E) and the Rep68-ssDNA (F) complexes at 20°C in buffer A, with Rep68 and DNA concentrations at 1 mg/ml and 2.8 µM, respectively.
Table 1.
Sedimentation coefficient and estimated molecular weights of the Rep68-RBA and Rep68-ssDNA complexes.
Figure 2.
Origin interaction domain (OID) is required but not sufficient for ssDNA-dependent Rep68.
Rep40 and OID proteins (16.6 µM) were incubated in the absence (A and D) or presence of 2.8 (B and E) or 16.6 µM ssDNA (C and F). OID was also incubated in the presence of 2.8 (G) or 16.6 µM RBS dsDNA (H). Fifty µL of sample was chromatographed on a Superdex 200 10/300 GL column with a flow rate of 0.5 mL/min. Protein elution was followed by UV detection at 280 nm. Molecular weight standards are shown on top, and dashed line corresponds to the elution position of Rep protein alone.
Figure 3.
R107 is required for ssDNA-dependent Rep68.
Rep proteins (16.6 µM) were incubated in the absence (A, C, E, G, I) or presence of 2.8 µM ssDNA (B, D, F, H, J). Fifty µL of sample was chromatographed on a Superose 6 10/300 GL column with a flow rate of 0.5 mL/min. Protein elution was followed by UV detection at 280 nm. Molecular weight standards are shown on top, and V0 position is represented as dashed line in Rep68 panels. (A and B) non-tagged Rep68; (C and D) His-Rep68 WT; (E and F) His-Rep68 K404A; (G and H) His-Rep68 K406A; and (I and J) His-Rep68 R107A.
Figure 4.
Cryo-electron microscopy of ssDNA-dependent oligomeric Rep68 rings.
Rep68-ssDNa complex was purified by size-exclusion chromatography in buffer A, and central part of the peak was concentrated and used for further cryo-electron microscopy analysis. (A) Representative image of the ssDNA-Rep68 oligomer; ring-shaped end view are shown by arrowheads. Bar corresponds to 20 nm. (C) A representative class average of end views is shown; internal and external dimensions of the ring are shown in Angstroms. (B) The ssDNA-Rep68 oligomer was purified by size-exclusion chromatography in buffer A; central part of the peak was concentrated and mixed with n-octyl β-D-glucopyranoside just before cryo-EM analysis. Arrowheads indicate side views of the Rep68 oligomer. Bar corresponds to 20 nm. (D) A representative class of side views is shown. Dimensions in Angstrom are shown for the length and width of the oligomer. (E and F) Two-dimensional projections of a double-octameric Rep68; end view (E), and side view (F).
Figure 5.
Rep68 forms an active helicase oligomer in the presence of ss-dsDNA heteroduplex.
Rep68 (1 mg/ml, 16.6 µM) was incubated with 2.8 µM ssDNA (A), 2.8 µM heteroduplex (B), or 2.8 µM Cy5-heteroduplex (C) on ice for 30 min. After centrifugation, 50-µL of sample was chromatographed on a Superose 6 HR 10/30 column. Elution of Rep68 oligomers was followed by UV detection at 280 nm. V0 position is represented as dashed line in Rep68 panels. (D) Size-exclusion chromatography of Rep-heteroduplex in buffer A. Central part of the high-MW peak (dashed lines) was concentrated, and mixed with n-octyl β-D-glucopyranoside just before cryo-EM analysis. (E and F) Representative images of the heteroduplex-Rep68 oligomer; ring-shaped and elongated particles are shown by arrowheads. Bars correspond to 20 nm. (G) A representative class average of end views is shown; internal and external dimensions of the ring are shown in Angstroms. (H) Rep68 (16.6 µM) was incubated with 2.1 µM Cy5-heteroduplex, and chromatographed as above. Three hundred-µL fractions were collected, and fraction corresponding to the central part of the Cy5-hetroduplex/Rep68 oligomer (in gray) was 4-fold concentrated. (I) Concentrated oligomer was incubated in the absence (−) or presence (+) of 1 mM MgCl2/1 mM ATP for 30 min at 37°C. Helicase reactions were analyzed on 16% polyacrylamide gels combined with Cy5 detection. −C and +C correspond to the negative (substrate alone) and positive (substrate plus Rep68) controls respectively, which were incubated in the presence of 1 mM MgCl2/1 mM ATP. M, Cy5-ssDNA marker (oligo JM-37). At right, positions for Cy5-heteroduplex and Cy5-ssDNA are indicated.