1. Volume 21, Issue 6, Pages 837-848 (March 2006)
Condensin I Interacts with the PARP-1-XRCC1 Complex and Functions in DNA Single- Strand Break Repair 
Jason T. Heale, Alexander R. Ball, John A. Schmiesing, Jong-Soo Kim, Xiangduo Kong, Sharleen Zhou, Damien F. Hudson, William C. Earnshaw, Kyoko Yokomori 
Molecular Cell 
Volume 21, Issue 6, Pages (March 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Identification of a 120 kDa Protein that Interacts with the NCTD of CNAP1
(A) The CNAP1 nuclear- and chromosome-targeting domain (NCTD) binds to a 120 kDa species (p120) in crude nuclear extracts. Far Western analysis of HeLa nuclear extracts (N.E.) and cytoplasmic extracts (C.E.) using a GST-GFP-NCTD (GG-NCTD) fusion protein as a radiolabeled probe. GST-GFP (GG) was used as a negative control. p120 is indicated by arrowhead. Coomassie stain of N.E. is also shown. A schematic diagram of the CNAP1-NCTD is shown underneath.
(B) Purification scheme for the isolation of p120 from HeLa N.E.
(C) Analysis of the purified p120. (Lanes 1–9) Far Western analysis of input, flowthrough (FT), and the final Q-column fractions. (Lanes 10–20) Silver staining of the same fractions showing the highly purified p120. (Lanes 21–30) Western blot analysis of the same fractions with anti-PARP-1 antibody. An arrowhead indicates p120 (PARP-1), while an asterisk indicates a PARP-1 cleavage product. Fraction numbers are shown at top.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
PARP-1 Interacts with Condensin I In Vivo in a Nuclear and S/G2 Phase-Specific Manner
(A) In vivo CoIP analysis of condensin interaction with PARP-1 using HeLa N.E. CoIPs with antibodies specific for condensin components CNAP1 and hCAP-G, cohesin component hSMC1, and preimmune IgG are indicated. Precipitates were eluted in two sequential steps using 1 M KCl and 2 M guanidine-HCl (2 M Guan). Eluted proteins from each step were TCA precipitated and analyzed by Western blotting using anti-PARP-1 antibody. Immunoprecipitated antigens are shown underneath.
(B) Reciprocal CoIP Western analysis using several anti-PARP-1 antibodies, followed by an anti-CNAP1 Western analysis. Goat polyclonal, mouse monoclonal, and rabbit polyclonal antibodies against PARP-1 were used for CoIP. Preimmune IgG served as a control.
(C) Nuclear-specific interaction of condensin and PARP-1. CoIP by anti-CNAP1 was performed using C.E. and N.E. Precipitates were analyzed by Western analysis for the presence of PARP-1. CoIP efficiency was assessed by anti-hCAP-G Western blot as shown underneath.
(D) The EtBr-resistant interaction between condensin I and PARP-1. CoIP of control (−) or EtBr treated (+) N.E. was carried out using an anti-hCAP-G antibody. Western blot detection of PARP-1 (arrowhead) and hCAP-G (bottom) are indicated.
(E) The condensin I-PARP-1 interaction peaks at S phase. HeLa cells were synchronized to different cell cycle stages (Figure S2A). N.E. were immunoprecipitated with anti-CNAP1 (lanes 2–4), preimmune IgG (lanes 5–7), or anti-hCAP-G antibodies (lanes 8–10) and analyzed by Western blotting with anti-PARP-1 antibody. An anti-hCAP-G Western blot indicates comparable precipitations of condensin I at different cell cycle stages.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Treatment of Cells with Genotoxic Agents Stimulates the Interaction between PARP-1 and Condensin I
After treatment of cells with HU (A) or MMS (B), nuclear extracts were immunoprecipitated with anti-hCAP-G antibody and analyzed by Western blot using anti-PARP-1 antibody. To ensure equal IP efficiency, the 2 M Guan fractions were also probed with anti-hCAP-H or CNAP1 antibody as indicated.
(A) Cells were treated for 24 hr with different concentrations of HU.
(B) Cells were harvested immediately following exposure to either 10 mM or 30 mM of MMS for 30 min or 1 hr as indicated.
(C) Extracts from cells treated with 20 mM H2O2 for 20 min were used in an anti-hCAP-E CoIP followed by an anti-PARP-1 Western blot.
(D) The PARP-1-condensin I interaction is induced throughout interphase in response to damage. HeLa cells synchronized to G1 or S/G2 phases were treated with H2O2 and harvested immediately. CoIP with anti-hCAP-G antibody was followed by anti-PARP-1 Western analysis. The 2 M Guan fractions were reprobed for hCAP-E to show IP efficiency.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Analysis of Condensin I Interactions with BER Factors
(A) CoIP of N.E. from H2O2-treated cells with anti-hCAP-G antibody probed with antibodies against PARP-1, XRCC1, FEN-1, or DNA ligase III as indicated. Cells were exposed to 20 mM H2O2 for 20 min and were either allowed no recovery (0 hr) or 1 hr of recovery (1 hr) as indicated. Control untreated HeLa cells (Cont) were harvested at the same time. Beads were probed with guinea pig anti-hCAP-G antibody to show IP efficiency.
(B) Similar CoIP in the absence (Cont) or presence of damage (H2O2) as in (A) followed by Western analysis using anti-DNA polymerase δ, ɛ, or β antibodies.
(C) Anti-hCAP-G CoIP of N.E. from H2O2-exposed HeLa treated with EtBr or micrococcal nuclease (MNase). Western blots were performed using antibodies specific for PARP-1 and XRCC1. Beads were probed for hCAP-G.
(D) The association between PARP-1 and XRCC1 increases upon damage induction. Anti-PARP-1 (polyclonal) CoIP of H2O2-exposed HeLa N.E. followed by anti-XRCC1 Western blot. Lanes 7 and 8 (beads) were reprobed with monoclonal PARP-1 antibody to show IP efficiency.
(E) Condensin association with XRCC1 is mediated through PARP-1. Nuclear extracts from wild-type (PARP+/+) and PARP-1 knockout (PARP−/−) MEFs treated with 20 mM H2O2 for 20 min were immunoprecipitated with anti-hCAP-G antibody and probed for XRCC1 by Western blotting. A CNAP1 Western analysis shows IP efficiency.
(F) Western analysis of sucrose gradient ultracentrifugation of N.E. from control or H2O2-treated HeLa cells. Fraction numbers from the bottom to the top of the gradient (1–12) and the fraction corresponding to 13S (arrowhead at the top) are indicated. Western blot analysis was done using antibodies against hCAP-G, PARP-1, XRCC1, FEN1, or ligase III as indicated.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Condensin I Interacts with Hypo-(ADP-ribosyl)ated PARP-1 and Associates with Damaged Chromatin
(A) Detection of auto-(ADP-ribosyl)ation of PARP-1 following H2O2 treatment. PARP-1 immunopurified using anti-PARP-1 polyclonal antibody from cells untreated (−) or treated (+) with H2O2 was analyzed by Western blot using monoclonal anti-PAR and anti-PARP-1 antibodies. Arrowhead indicates hypo-(ADP-ribosyl)ated form of PARP-1.
(B) Condensin I interacts with a non- or hypo-(ADP-ribosyl)ated form of PARP-1. Nuclear extracts from cells untreated (−) or treated (+) with H2O2 were used in anti-hCAP-E CoIPs. Precipitates were eluted in 2 M Guan after a 1 M KCl wash and probed with monoclonal anti-PARP-1 (lanes 1–4) and anti-PAR (lanes 5–8) antibodies to determine the ADP-(ribosyl)ation status of PARP-1.
(C) Sequential extraction of HeLa cells without and with H2O2 treatment. CSK and EXT fractions contain cytoplasmic, cytoskeletal, and soluble nuclear proteins. Insoluble nuclear pellet fractions contain proteins that are tightly bound to chromatin and/or the nuclear matrix. The blot was probed for PARP-1, XRCC1, the five condensin I subunits, and the cohesin subunit RAD21 (loading control). The apparent decrease of these proteins (except for RAD21) in the CSK fraction reflects the decrease of soluble nuclear proteins, which now associate tightly with the chromatin-enriched nuclear pellet.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Condensin-Depleted Cells Are Compromised in SSB Repair
(A) An alkaline comet assay performed on ScII (CAP-E)-depleted chicken DT40 cells. Parental wild-type cells treated with Dox (ScII ON), and ScII conditional knockout cells without Dox (ScII ON) or with Dox (ScII OFF), were exposed to 150 μM H2O2 for 10 min and allowed no recovery (immediate) or 45 min to 2 hr of recovery. Tail moment was used as a relative indicator of severity of damage within a cell. A Western blot shows the efficient depletion of ScII following a 36 hr exposure to Dox. A β-tubulin Western blot was used as a loading control.
(B) An alkaline comet assay on HeLa cells treated with siRNA against CNAP1. Following depletion of CNAP1, cells were treated as described in (A) and analyzed immediately, 1 hr and 2 hr after damage induction by comet assay. A Western blot demonstrates depletion of CNAP1 in siRNA-treated cells compared to mock and control siRNA (nonsilencing) treated cells. A Western analysis of cohesin component Rad21 was used as a loading control.
(C) A neutral comet assay to detect DSBs following IR. The siRNA-treated HeLa cells were exposed to 10 Gy γ irradiation and analyzed as in (B).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 A Model for the Recruitment of Condensin I to SSB Sites
SSB damage induces complex formation between condensin I, PARP-1, and XRCC1. This complex is recruited to the DNA lesion and interacts with additional BER factors such as FEN-1 and DNA polymerases δ and ɛ. Condensin I may be involved in local chromatin/DNA structural organization that allows efficient BER.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions