Studies with the human cohesin establishment factor, ChlR1. Association of ChlR1 with Ctf18-RFC and Fen1

Abstract

Human ChlR1 (hChlR1), a member of the DEAD/DEAH subfamily of helicases, was shown to interact with components of the cohesin complex and play a role in sister chromatid cohesion. In order to study the biochemical and biological properties of hChlR1, we purified the protein from 293 cells and demonstrated that hChlR1 possesses DNA-dependent ATPase and helicase activities. This helicase translocates on single-stranded DNA in the 5' to 3' direction in the presence of ATP and, to a lesser extent, dATP. Its unwinding activity requires a 5'-singlestranded region for helicase loading, since flush-ended duplex structures do not support unwinding. The helicase activity of hChlR1 is capable of displacing duplex regions up to 100 bp, which can be extended to 500 bp by RPA or the cohesion establishment factor, the Ctf18-RFC (replication factor C) complex. We show that hChlR1 interacts with the hCtf18-RFC complex, human proliferating cell nuclear antigen, and hFen1. The interactions between Fen1 and hChlR1 stimulate the flap endonuclease activity of Fen1. Selective depletion of either hChlR1 or Fen1 by targeted small interfering RNA treatment results in the precocious separation of sister chromatids. These findings are consistent with a role of hChlR1 in the establishment of sister chromatid cohesion and suggest that its action may contribute to lagging strand processing events important in cohesion.

FIGURE 1.

Purification of recombinant hChlR1; examination of its helicase and ATPase activities. Wild-type and mutant hChlR1 were overexpressed and purified from 293 cells as described under “Experimental Procedures.” A, left, Coomassie-stained SDS-polyacrylamide gel of purified proteins (4 and 2 μg of hChlR1 and KRm, respectively); right, Western blot analysis of SDS-polyacrylamide gel using affinity-purified Hel1 antisera that recognizes the N terminus of the hChlR1 protein (200 ng of hChlR1 and KRm). B, cosedimentation of hChlR1 protein, DNA helicase, and DNA-dependent ATPase activities. Top,50 μg of wild type hChlR1 protein was loaded onto a 5-ml 15–40% glycerol gradient; after centrifugation for 20 h at 250,000 × g, aliquots (2 μl) of the eluted fractions were assayed for helicase activity (middle) using the 18mer-M13 helicase substrate; DNA-dependent ATPase activity (bottom). BSA, bovine serum albumin.

FIGURE 2.

Properties of hChlR1 helicase activity. Helicase assays were carried out using conditions described under “Experimental Procedures.” All reactions contained 36 fmol of hChlR1 and 5 fmol of the 39mer-M13 substrate, unless specified. A, nucleotide requirement for hChlR1 helicase activity. Each reaction contained, where noted, a 1 mm concentration of the indicated nucleoside 5′-triphosphate. B, unwinding of the 18mer-M13 or 39mer-M13 helicase substrate (18- and 39-mer, respectively) in the presence of increasing levels of hChlR1 and 1 mm ATP or dATP. C, the influence of various cations on hChlR1 helicase activity.

FIGURE 3.

Characterization of DNA-dependent ATPase activity of hChlR1. Rate of hydrolysis of ATP and dATP by hChlR1. Reactions were carried out as described under “Experimental Procedures” in the presence of 90 fmol of hChlR1 and 40 fmol of the 18-mer M13 substrate.

FIGURE 4.

Helicase activity of hChlR1 with various DNA substrates. DNA helicase reactions were carried out as described under “Experimental Procedures” with increasing amounts of hChlR1 and 5 fmol of the indicated partial duplex DNA substrates. In all panels shown, the conditions used were as follows. Lane 1, substrate only; lane 2, boiled substrate; lanes 3–5, 1, 3, or 9 fmol of enzyme, respectively; lane 6, 9 fmol of enzyme but no ATP. A, hChlR1 helicase translocates in the 5′–3′ direction. B, displacement of nicked and gapped DNA substrates by hChlR1. The size of the single strand gap separating the two labeled oligonucleotide is noted above the panels. The sequences of oligonucleotides used to generate the substrates are noted, and their sequences are summarized in Table 1.

FIGURE 5.

A, length of duplex unwound by hChlR1 is stimulated by RPA. DNA helicase activity was assayed using 5 fmol of the partial duplex M13 substrate (39mer40dT-M13, 5 fmol) containing duplex regions varying in length between 40 and 600 bp and a 40-nt oligo(dT) tail at its 5′-end. The indicated levels of hChlR1 were preincubated with the substrate in the standard helicase reaction but in the presence of 100 μm ATP, at 25 °C for 10 min, after which human RPA (0.32 pmol) was added as indicated, and all reactions were adjusted to 1 mm ATP. After incubation at 37 °C for 30 min, reactions were stopped by the addition of 20 mm EDTA, 0.1% SDS, 0.125 mg/ml proteinase K, and the mixture were incubated for an additional 30 min at 37 °C and then subjected to electrophoretic separation through a neutral 1% agarose gel. Lane 1, ³²P-labeled 100-bp ladder DNA marker (denatured); lane 2, boiled substrate; lanes 3 and 10, reactions without hChlR1; lanes 4–6 and 7–9, increasing amounts of hChlR1 in the absence or presence of RPA. B, length of duplex unwound by hChlR1 is stimulated by Ctf18-RFC. The hChlR1-catalyzed displacement reaction was carried out as described in A, with hChlR1 preincubation and the subsequent addition of Ctf18-RFC, RFC, or PCNA, as indicated.

FIGURE 6.

hChlR1 interacts with the Ctf18-RFC complex and PCNA. A, in the three upper panels, 1 m g of lysates from 293 cells transiently expressing FLAG-Ctf18 (second panel from top) or FLAG-Dcc1 (third panel from top) alone (lane 2) or together with constitutively expressed ChlR1-HA (lane 4) were incubated with HA antibody as indicated at the top of the immunoblots; specific interactions were detected by Western blotting, using antibodies to hChlR1 (top panel), Ctf18 (second panel from top), or Dcc1 (third panel from top). In the bottom panel, lysates from 293 cells (lanes 2) or 293 cells constitutively expressing ChlR1-HA (lanes 4) were incubated with HA antibody, and specific interactions between ChlR1-HA and endogenous p37/RFC2 subunit were detected by Western blotting using p37/RFC2 antibody. B, reciprocal immunoprecipitations in 293 cells expressing both ChlR1-HA and FLAG-tagged Ctf18 performed with protein A beads in combination with antibodies specific for Ctf18 (lane 2) or with preimmune serum (lane 3), as indicated at the top of the immunoblot; specific interactions were detected by Western blotting using antibodies to hChlR1 (top) and Ctf18 (bottom). C, immunoprecipitation using FLAG beads of lysates from 293 cells transiently expressing ChlR1-HA alone (lane 4) or together with FLAG-tagged Dcc1 (lane 3); specific interactions were detected by Western blotting using antibodies to hChlR1 (top) and Dcc1 (bottom). D, hChlR1 does not interact with RFC1. Lysates from 293 cells constitutively expressing ChlR1-HA (lane 3) were incubated with HA antibody; no specific interaction was detected by Western blotting using antibodies to hChlR1 (top) and the RFC1 subunit (bottom). E, hChlR1 interacts with PCNA. Left, 1 mg of 293 cell lysates transiently expressing FLAG-ChlR1 (lane 2) or 293 cell lysates (lane 3), supplemented with recombinant HA-PCNA (5.6 pmol) were incubated with FLAG-M2 antibody beads; right, 1 mg of 293 cell lysates transiently expressing FLAG-ChlR1 in the presence (lane 5) or absence (lane 6) of recombinant HA-PCNA (5.6 pmol) were incubated with HA antibody beads; specific interactions were detected by Western blotting using antibodies to hChlR1 (top) and PCNA (bottom). In all immunoprecipitations, input represents 10% of the total amount of lysate/recombinant protein used for immunoprecipitation. IP, immunoprecipitation.

FIGURE 7.

hChlR1 interacts with Fen1 and stimulates its endonuclease activity. A, 1 mg of lysates from 293 cells transiently expressing FLAG-Fen1 alone (lane 2) or together with constitutively expressed ChlR1-HA (lane 4) were incubated with HA antibody as indicated at the top of the immunoblots; specific interactions were detected by Western blotting using antibodies to hChlR1 (top) and Fen1 (bottom). B, reciprocal immunoprecipitations in 293 cells expressing ChlR1-HA and FLAG-tagged Fen1 performed with protein A beads in combination with Fen1 antibodies (lane 2) or preimmune serum (lane 3), as noted at the top of the immunoblot; specific interactions were detected by Western blotting using antibodies to hChlR1 (top), and Fen1 (bottom). C, hChlR1 directly interacts with Fen1. Recombinant His-tagged-Fen1 (5.6 pmol) and recombinant FLAG-tagged ChlR1 (5 pmol) were incubated with FLAG-M2 antibody beads (lanes 3 and 4); proteins were eluted in the absence (lane 3) or presence (lane 4) of 1 mg/ml FLAG peptide, and specific interactions were detected by Western blotting using antibodies specific to hChlR1 (top) and Fen1 (bottom). Input represents 10% of total amount of lysate/recombinant protein used for immunoprecipitation. D, hChlR1 stimulates Fen1 endonuclease activity. Fen1 (5 fmol) was incubated in the absence (lanes 2–5) or presence (lanes 6–9) of the indicated amounts of RPA with or without hChlR1 (lanes 7–9 and lanes 3–5, respectively) in standard flap endonuclease reaction mixtures (described in Ref. 36) in the presence of the 729/5TBG substrate (described in Table 1), whose structure is shown on the left. IP, immunoprecipitation.

FIGURE 8.

siRNA-mediated depletion of hChlR1, Fen1, or Scc1 leads to defects in sister chromatid pairing. A, HeLa cells were transfected with control (aspecific siRNA) or hChlR1-, Fen1-, or Scc1-specific siRNAs and metaphase spreads prepared 48 h following transfection. DNA was stained with 4′,6-diamidino-2-phenylindole, whereas the inset shown to the right represents a higher magnification of a sister chromatid pair; the size reference bar shown is 3 μm long. B (top), the centromeric region of chromosome 9 was probed using the FISH procedure (33), DNA was stained with 4′,6-diamidino-2-phenylindole, and chromosomes were visualized by microscopy; the size reference bar shown is 1 μm. Bottom, the distance between chromatid pairs of at least 100 chromosomes in each case was measured using MetaMorph software and grouped together by the distance separating the sister chromatid as shown in the lower right side of the figure. The results are expressed as the mean percentage of cells ± S.D. of the experiment performed in triplicate. C, an aliquot of cells from each siRNA experiment was harvested and subjected to Western blot analysis to determine the expression of hChlR1, Fen1, and Scc1 proteins using antibodies specific for each protein.α-Tubulin was included as loading control.