Dual roles of the 11S regulatory subcomplex in condensin functions - PubMed (original) (raw)

Dual roles of the 11S regulatory subcomplex in condensin functions

K Kimura et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Condensin is a multisubunit protein complex that reconfigures DNA structure in an ATP-dependent manner in vitro and plays a central role in mitotic chromosome condensation in Xenopus egg cell-free extracts. The Xenopus 13S condensin complex (13SC) is composed of two subcomplexes: an 8S core subcomplex (8SC) consisting of two structural maintenance of chromosomes (SMC) subunits (XCAP-C and -E) and an 11S regulatory subcomplex (11SR) containing three non-SMC subunits (XCAP-D2, -G, and -H). We report here the biochemical and functional dissection of this chromosome condensation machinery. Although both 8SC and 13SC can bind to DNA in vitro and contain the SMC ATPase subunits, only 13SC is active as a DNA-stimulated ATPase and supports ATP-dependent supercoiling activity. In the cell-free extracts, 13SC is the active form that binds to chromosomes and induces their condensation. Neither 11SR nor 8SC alone is able to bind to chromatin. Our results suggest that the non-SMC subunits have dual roles in the regulation of condensin functions: one is to activate SMC ATPases and the other is to allow the holocomplex to associate with chromatin in a mitosis-specific manner.

PubMed Disclaimer

Figures

Figure 1

Figure 1

A diagram for sequential affinity-purification of the holocomplex and subcomplexes of condensin. See the text for details.

Figure 2

Figure 2

Characterization of purified complexes. (A) Affinity-purified fractions of 11SR (lanes 1 and 4), 13SC (lanes 2 and 5), and 8SC (lanes 3 and 6) were subjected to SDS/7.5% PAGE, and stained with Coomassie blue (lanes 1–3) or analyzed by immunoblotting with antibodies against condensin subunits (lanes 4–6). (B) An affinity-purified fraction of 11SR (Top), 13SC (Middle), or 8SC (Bottom) was fractionated in a 5–20% sucrose gradient. Fractions were resolved by SDS/7.5% PAGE and analyzed by immunoblotting.

Figure 3

Figure 3

Biochemical activities of condensin and its subcomplexes. (A) DNA-coupled (lanes 2, 4, and 6) or control (lanes 1, 3, and 5) paramagnetic beads were incubated with the same concentration of purified 11SR (Top), 13SC (Middle), or 8SC (Bottom) in a buffer containing 50 mM KCl (lanes 1 and 2), 100 mM KCl (lanes 3 and 4), or 400 mM KCl (lanes 5 and 6) at 22°C for 1 h. After washing the beads with the same buffer, bound proteins were analyzed by immunoblotting. As standards, 6.3% (lane 7), 12.5% (lane 8), 25% (lane 9), and 50% (lane 10) of input proteins were loaded. (B) Increasing amounts of 11SR, 13SC, or 8SC were incubated with a relaxed circular DNA in the presence of calf thymus topoisomerase I and 1 mM Mg-ATP in a buffer containing 50 mM KCl. DNA was purified, electrophoresed in a 0.7% agarose gel, and visualized by Southern blotting. The positions of relaxed DNA (r) and ladder of supercoiled DNA (s) are indicated. The molar ratios of complex to DNA present in the reaction mixtures were 0 (lanes 1, 5, and 9), 22.5:1 (lanes 2, 6, and 10), 45:1 (lanes 3, 7, and 11), and 90:1 (lanes 4, 8, and 12). (C) An affinity-purified fraction of 11SR (columns 1 and 5), 13SC (columns 2 and 6), 8SC (columns 3 and 7), or buffer alone (columns 4 and 8) was assayed for ATPase activity without DNA (open bar), or with double-stranded DNA (filled bar) at 50 mM KCl or 100 mM KCl. ATP and its hydrolysis product ADP were separated by TLC and quantitated (17). The activities are shown as pmol of hydrolyzed ATP/min⋅pmol of holocomplex or subcomplex.

Figure 4

Figure 4

Immunodepletion and rescue. (A) Mitotic extracts were immunodepleted with control IgG (lane 1), with a mixture of anti-XCAP-C, -D2, -E, and -G (lanes 2–5), or with a mixture of anti-XCAP-D2 and -G (lanes 6 and 7). Then a purified fraction of 13SC (lane 3), 8SC (lane 4), 11SR (lanes 5 and 7), or buffer (lanes 2 and 6) was added back into the depleted extracts. Aliquots of each extract were analyzed by immunoblotting. (B) After incubating sperm chromatin with each extract at 22°C for 2 h, chromatin was isolated and bound proteins were analyzed by immunoblotting. (C) Sperm chromatin was incubated with each extract (1–7 as in A) at 22°C for 2 h, fixed, and stained with 4′,6-diamidino-2-phenylindole. (Bar, 10 μm.)

Figure 5

Figure 5

Cell cycle regulation of interactions between 13SC and nucleosomes. (A) DNA-coupled magnetic beads were incubated with an interphase extract (lanes 1–3) or a mitotic extract (lanes 4–6) for different times as indicated. Alternatively, the DNA beads were first incubated with an interphase extract for 60 min and then with a mitotic extract for the indicated times (lanes 7–9). After washing the beads, bound proteins were analyzed by immunoblotting. (B) I nucleosomes, M nucleosomes, and I→M nucleosomes were assembled as shown in A. After washing the beads, DNA was digested with increasing amounts of micrococcal nuclease, isolated, and fractionated in a 1.25% agarose gel. m, 100-bp ladder marker. (C) DNA-coupled magnetic beads (lanes 4–10) or control beads (lanes 1 and 2) were incubated with buffer alone (lanes 1–4), with a condensin-depleted interphase extract for 120 min (lanes 5, 7, and 9), or with a condensin-depleted interphase extract for 60 min and then with a condensin-depleted mitotic extract for another 60 min (lanes 6, 8, and 10). The beads were washed and incubated with the mitotic form of 13SC (lanes 1, 3, 7, and 8), with the interphase form of 13SC (lanes 2, 4, 9, and 10), or with buffer alone (lanes 5 and 6) for 60 min. After washing the beads, bound 13SC was detected by immunoblotting. As standards, 6.25% (lane 11), 12.5% (lane 12), 25% (lane 13), and 50% (lane 14) of input 13SC were loaded in parallel. (D) Condensin-free I nucleosomes (lanes 1, 2, and 4) or I→M nucleosomes (lanes 3, 5, and 6) were assembled around DNA-coupled beads as described in C. The beads were washed and treated with protein kinase A (lane 2), λ phosphatase (lane 6), or control buffers (lanes 1 and 3–5). The phosphorylation states of histone H3 were analyzed by immunoblotting with a phosphospecific antibody that recognizes Ser-10 (Upper). Alternatively, the beads were incubated with the mitotic form of 13SC for 60 min. After washing the beads, bound proteins were analyzed by immunoblotting (Lower).

Similar articles

Cited by

References

    1. Koshland D, Strunnikov A. Annu Rev Cell Dev Biol. 1996;12:305–333. - PubMed
    1. Hirano T. Annu Rev Biochem. 2000;69:115–144. - PubMed
    1. Hirano T, Mitchson T J. Cell. 1994;79:449–458. - PubMed
    1. Hirano T, Kobayashi R, Hirano M. Cell. 1997;89:511–521. - PubMed
    1. Strunnikov A V, Larionov V L, Koshland D. J Cell Biol. 1993;123:1635–1648. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources