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. 2004 Jun 21;165(6):801-12.
doi: 10.1083/jcb.200402095. Epub 2004 Jun 14.

Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases

Wenhui Li  1 , Soo-Mi Kim, Joon Lee, William G Dunphy
Affiliations

Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases

Wenhui Li et al. J Cell Biol. .

Abstract

Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIalpha (Xtop3alpha) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3alpha has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.

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Figures

Figure 1.
Figure 1.
Xblm binds to chromatin and becomes phosphorylated in a checkpoint-dependent manner. (A) Extracts were incubated with no sperm chromatin (lane 1), untreated sperm chromatin (lane 2), sperm chromatin plus 100 μg/ml aphidicolin (APH; lane 3), UV-damaged sperm chromatin (lane 4), or sperm chromatin plus aphidicolin and caffeine (lane 5). After 90 min, nuclei were isolated and separated into nuclear soluble and chromatin fractions. The fractions were immunoblotted for Xblm and Xorc2 (as a loading control). (B) Extracts were incubated for 90 min with untreated sperm chromatin (lane 1), UV-damaged sperm chromatin (lane 2), or sperm chromatin plus aphidicolin (lane 3). Whole nuclear fractions were immunoblotted for Xblm. (C) Extracts were incubated with sperm chromatin alone (lane 1), sperm chromatin plus aphidicolin (lane 2), or sperm chromatin plus aphidicolin and caffeine (lane 3). Nuclear fractions were immunoblotted for Xblm. (D) Anti-Xblm immunoprecipitates from extracts containing no DNA (lane 1) or pA-pT (lanes 2 and 3) were incubated without (lanes 1 and 2) or with (lane 3) λ phosphatase and were immunoblotted for Xblm.
Figure 2.
Figure 2.
Xatr and Xrad17, but not Claspin, are required for phosphorylation of Xblm. (A) Extracts were treated with control (lanes 1 and 2), anti-Xatr (lanes 3 and 4), or anti-Claspin (lanes 5 and 6) antibodies. Sperm chromatin was incubated in extracts for 90 min in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of aphidicolin. Nuclear fractions were isolated and immunoblotted for Xatr, Claspin, Xblm, Xchk1, and P-Ser344 of Xchk1. (B) Extracts were treated with control (lanes 1, 2, 5, and 6) or anti-Xrad17 (lanes 3, 4, 7, and 8) antibodies. Sperm chromatin was incubated in extracts for 90 min in the absence (lanes 1 and 5) or presence (lanes 2–4 and 6–8) of aphidicolin. His6-Xrad17 was added back to some extracts (lanes 4 and 8). Chromatin fractions and whole-extract aliquots were immunoblotted for Xatr, Xblm, and Xrad17.
Figure 3.
Figure 3.
Xblm is not required for the checkpoint-dependent phosphorylation of Xchk1 or Xchk2. (A) Untreated (lanes 1 and 2), Xblm-depleted (lanes 3 and 4), and mock-depleted (lanes 5 and 6) egg extracts containing 35S-labeled Xchk1 were incubated for 90 min in the absence or presence of aphidicolin. Nuclear fractions were isolated and immunoblotted for Xblm or were examined for phosphorylation of 35S-labeled Xchk1 by SDS-PAGE and phosphorimaging. (B) Extracts were treated with control antibodies (lanes 1 and 2), anti-Xblm antibodies (lanes 3 and 4), or no antibodies (lanes 5 and 6). 35S-labeled Xchk2 was incubated in these depleted extracts for 90 min in the absence or presence of pA-pT. 35S-labeled Xchk2 in the whole-egg extracts was examined by phosphorimaging. (C) Extracts were treated with control antibodies (lanes 1 and 2), anti-Xrad17 antibodies (lane 3), or both anti-Xrad17 and anti-Xblm antibodies (lane 4). Extracts were incubated with sperm chromatin in the absence (lane 1) or presence (lanes 2–4) of aphidicolin. Chromatin fractions were immunoblotted for Xatr, Xblm, and Xrad17. Phosphorylation of Xchk1 in the soluble nuclear fraction was detected with anti-P-Ser344 antibodies. (D) Extracts were treated with control (lanes 1 and 2), anti-Xrad17 (lane 3), or both anti-Xrad17 and anti-Xblm (lane 4) antibodies. Phosphorylation of 35S-labeled Xchk2 after incubation in the absence (lane 1) or presence (lanes 2–4) of pA-pT in egg extracts was examined.
Figure 4.
Figure 4.
Xblm interacts with Xtop3α in a regulated manner. (A) Control (lane 1) and anti-Xblm (lanes 2 and 3) antibodies on protein A beads were incubated in extracts in the absence (lanes 1 and 2) or presence (lane 3) of pA-pT. The beads were collected and immunoblotted for Xblm and Xtop3α. (B) Control antibodies (lanes 1 and 4) and two different anti-Xblm antibodies (#870, lanes 2 and 5; #868, lanes 3 and 6) on protein A beads were incubated in extracts in the absence (lanes1–3) or presence (lanes 4–6) of pA-pT. The beads were collected and immunoblotted for Xtop3α. (C) His6-tagged Xblm-N122 on nickel beads and blank nickel beads were incubated in interphase egg extracts. The beads were collected and immunoblotted for Xtop3α. (D) Xblm-N122 on nickel beads was incubated in extracts containing no DNA (lane 1), pA-pT (lane 2), or pA-pT plus caffeine (lane 3). The beads were collected and immunoblotted with anti-Xtop3α and anti-His6 antibodies (to detect Xblm-N122). (E) Egg extracts were incubated with no DNA (lane 1), pA-pT (lane 2), or pA-pT plus caffeine (lane 3) and were immunoblotted for Xblm. (F) Anti-Xblm antibodies on protein A beads were incubated in extracts in the presence of different oligonucleotides. In some cases, either purified Xblm-N122 (lanes 6–10) or caffeine (lanes 11–14) was also added. After 2 h, the beads were collected and immunoblotted for Xblm and Xtop3α.
Figure 5.
Figure 5.
The NH2-terminal 122 amino acids of Xblm are involved in the interaction with Xtop3α on chromatin. (A) Extracts were treated with no antibodies (lanes 1–3), anti-Xblm antibodies (lanes 4 and 5), or control antibodies (lanes 6 and 7) and then incubated with sperm chromatin alone (lanes 1, 4, and 6), sperm chromatin plus aphidicolin (lanes 2, 5, and 7), or sperm chromatin plus aphidicolin and caffeine (lane 3). After 90 min, nuclear fractions were isolated and separated into chromatin and nuclear soluble fractions. These fractions were immunoblotted for Xblm, Xtop3α, and Xorc2. (B) The NH2-terminal 122 amino acids of Xblm are required for interaction with Xtop3α on chromatin. Egg extracts containing sperm chromatin were incubated in the absence (lanes 1–4) or presence (lanes 5–8) of aphidicolin. The extracts were also treated without (lanes 1 and 5) or with (10–40 μg/μl; lanes 2–4 and 6–8) Xblm-N122. After 90 min, chromatin fractions were immunoblotted for Xblm, Xtop3α, and Xorc2. (C) Extracts were treated with no antibodies (lane 1), control antibodies (lane 2), or anti-Xtop3α antibodies (lane 3) and were immunoblotted for Xblm (top) and Xtop3α (bottom). (D) Mock-depleted extracts (lanes 1 and 2), Xtop3α- depleted extracts (lanes 3 and 4), and extracts treated with no antibodies (lanes 5 and 6) from C were incubated with sperm chromatin in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of aphidicolin. Chromatin fractions were isolated and immunoblotted for Xblm, Xtop3α, and Xorc2.
Figure 6.
Figure 6.
Phosphorylation of Xblm depends on association with Xtop3α. (A) Buffer alone (lanes 1 and 2) or Xblm-N122 (100 μg/μl; lanes 3 and 4) was added to extracts lacking (lanes 1 and 3) or containing (lanes 2 and 4) aphidicolin. After 90 min, nuclear fractions were isolated and immunoblotted with antibodies against Xblm and P-Ser344 of Xchk1. (B) Mock-depleted (lanes 1 and 2) and Xtop3α-depleted (lanes 3 and 4) extracts were incubated with sperm chromatin in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of aphidicolin. After 90 min, nuclear fractions were isolated and immunoblotted with antibodies against Xblm, Xtop3α, and the whole Xchk1 protein.
Figure 7.
Figure 7.
Xblm associates with replicating chromatin, but is not essential for DNA replication. (A) Sperm chromatin was incubated in egg extracts in the absence (lanes 1–5) or presence (lanes 6–10) of aphidicolin. At various times, chromatin fractions were isolated and immunoblotted for Xblm, RPA70, and Xorc2. (B) Xblm-depleted (lanes 1–4) and mock-depleted (lanes 5–8) egg extracts were incubated with sperm chromatin. Chromosomal DNA replication was assayed as described in the Materials and methods. The two bands in each lane depict 32P incorporation into chromosomal DNA. (C) Xblm-depleted (lanes 1 and 2) and mock-depleted (lanes 3 and 4) extracts were incubated with sperm chromatin in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of aphidicolin. Chromatin fractions were isolated and immunoblotted for Xblm, RPA70, and Xorc2. (D) Mock-depleted (lanes 1 and 2) and RPA70-depleted (lanes 3 and 4) extracts were incubated with sperm chromatin in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of aphidicolin. Chromatin fractions were isolated and immunoblotted for Xblm, RPA70, and Xorc2. (E) Extracts were left untreated (lane 1) or were treated with either geminin (lane 2) or p27 (lane 3) as described previously (Lee et al., 2003), and sperm chromatin was added. After 90 min, chromatin fractions were immunoblotted for Xblm (top) and Xorc2 (bottom).
Figure 8.
Figure 8.
Xblm is required for preventing accumulation of chromosomal breaks during normal and disrupted S phases. (A) Extracts were treated with control antibodies (lanes 1–4), anti-Xblm antibodies (lanes 5–8), or no antibodies (lanes 9–12) and were incubated with sperm chromatin. Some extracts were treated with 0.1 U/μl EcoRI (lanes 9–12) and geminin (lanes 3, 4, 7, 8, 11, and 12). After 2 h, TUNEL assays were performed in the presence (lanes 2, 4, 6, 8, 10, and 12) or absence (lanes 1, 3, 5, 7, 9, and 11) of TdT. Samples were subjected to agarose gel electrophoresis and phosphorimaging (top). The radioactivity in each band was quantitated (bottom). (B) Mock-depleted (lane 1) and Xblm-depleted (lanes 2 and 3) extracts were prepared. Recombinant Xblm was made in the TnT® system and added back to an Xblm-depleted extract (lane 3). Samples were immunoblotted for Xblm. (C) Extracts were treated with control antibodies (lanes 1 and 7), anti-Xblm antibodies (lanes 2, 3, 8, and 9), or no antibodies (lanes 4, 5, 6, and 10) and were incubated with sperm chromatin. The reactions also contained 100 μg/ml Xblm-N122 (lanes 4 and 10), 0.1 U/μl EcoRI (lane 6), and aphidicolin (lanes 7–10). Full-length recombinant Xblm was added to some samples (lanes 3 and 9). After 2 h, TUNEL assays were performed as in A.
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