Replication timing of the human 5q23/31.

<p><b>A.</b> Experimental strategy for determination of replication timing. Asynchronously replicating cells were labeled with BrdU and sorted by FACS into six fractions (G1, S1–4, G2/M) on the basis of DNA content. Genomic DNA from cells in each fraction was extracted, and newly replicated DNA was immunoprecipitated with anti-BrdU antibody. Semi-quantitative PCR was carried out using the newly replicated DNA as template. Relative band intensity was quantified. The values in each fraction were normalized by the levels of BrdU-labeled mitochondrial DNA (mtDNA; replicated equally throughout the cell cycle) used as an internal control for the recovery of DNA in each sample. <b>B.</b> 40,000 cells (Jurkat and HL-60) sorted (upper) and collected on the basis of DNA content (G1, S1–4, G2/M) were stained with PI, and analyzed by FACS (lower). <b>C.</b> Validation of cell cycle fractionation. The known early (PGK1) or late (F9) replicating region is enriched in appropriate fractions in comparison with the level of mtDNA in HL-60. <b>D.</b> DNA replication timing on the human chromosome 5q23/31 region (3.5 Mb) containing the cytokine cluster region in Jurkat (T cell) and HL-60 (non T cell). The 2.2 Mb segment containing the cytokine cluster (130.3–132.5) replicates in G1 or early in the S-phase (S1 and S2), whereas the 0.9 Mb segment distal to the cluster and proximal to the centromere replicates late in the S phase (S3, S4 and G2). The mean locations of the timing transition region (TTR) are located at around 130.15–130.25 (yellow box) in Jurkat (TTR-J) and at around 129.95–130.05 (green box) in HL-60 (TTR-H), and are offset by 180 kb in the two cell types. The left boundary of early replicating region coincided with the transition of chromosomal synteny (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042375#pone.0042375.s002" target="_blank">Fig. S2A</a></b>). <b>E.</b> The results of replication timing assays with fractionated cells are shown for each location along the 3.5 Mb human chromosome. The locations of the 9 primers used are indicated along the 5q23/31 region shown to the right of the panels. Small red solid boxes show the peak timing fraction for each probe, and large red dotted boxes show the maximum timing transition segments for Jurkat (129.98–130.22) and HL-60 (129.52–130.16).</p

Genome copy number analyses of TRR in synchronized cell population.

<p><b>A.</b> Locations of primers (a, b, c, d and f) used for copy number analyses. <b>B.</b> The procedure for quantification of copy number. <b>C</b>. Time course of DNA replication at various locations in and around TTR. Genome copy numbers of cells synchronously growing from double thymidine block were quantified at various loci at various timepoints. The level of DNA synthesis at 2 hr or 8 hr after release was set as 0% or 100% DNA synthesis, respectively.</p

Correlation between SATB1 expression and replication timing at TTR.

<p><b>A.</b> SATB1 expression in Jurkat, HL-60 and HeLaS3. Whole cell extracts, separated on 7.5% SDS-polyacrylamide gel, were blotted with anti-SATB1 antibody. Expression of SATB1 is high in T cell (Jurkat), and low or non-detectable in non-T cells (HL-60 and HeLaS3). <b>B.</b> Replication timing of the Probe 4 (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042375#pone.0042375.s002" target="_blank">Fig. 2B</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042375#pone.0042375.s006" target="_blank">Table S1</a></b>) in Jurkat, HL-60 and HeLaS3. This locus replicates in late-S in Jurkat (high SATB1) and in early-S in HL-60 (low SATB1) and HeLaS3 (very low SATB1).</p

Chromatin immunoprecipitation (ChIP) assays of SATB1 binding.

<p><b>A.</b> Locations of primers (a–e) used for ChIP assays. <b>B.</b> ChIP analyses were carried out by using anti-mouse SATB1 antibody (left panel) or purified mouse IgG1 control antibody (central panel) jn HeLaS3 cells stably expressing SATB1. Chromain-immunoprecipitated DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. Error bars represent the mean and standard deviations based on four independent experiments. Relative ratio (SATB1/control) is shown as SATB1-specific binding (right panel).</p

Effect of SATB1 expression on replication timing at TTR: suppression of SATB1 expression in Jurkat cells.

<p><b>A.</b> The procedure for repression of SATB1 expression in Jurkat. <b>B.</b> Whole cell extracts were examined by western blotting using anti-SATB1 antibody. Lane 1, untransfected Jurkat; lane 2, Jurkat transfected with pRS vector; lane 3 and 4, Jurkat transfected with pRS-SATB1-shRNA1 and with pRS-SATB1-shRNA1+ pRS-SATB1-shRNA2, respectively. Cells were harvested at 72 hr after transfection. <b>C.</b> Replication timing was determined by FISH across the TTR. Only the data at the Probe 4 are shown. Replication timing in the transition region changed from late (Jurkat) to early (SATB1-depleted Jurkat) (indicated by the arrows). At least 200 BrdU-positive nuclei (S-phase) were counted.</p

Analyses of replication timing by FISH.

<p><b>A.</b> Hybridization signals of replicating cells. SS, singlet-singlet: SD, singlet-doublet; DD, doublet-doublet. <b>B.</b> Replication timing analyzed by FISH at 5q23/31. Locations of DNA probes derived from 5q23/31used in this study are shown at the top. Human BAC clones were purchased from Invitrogen. A cosmid clone on the chromosome 12, cCl12–140, was kindly provided by Dr. Okumura (Nogami et al, 2000), and was used as a control for early replication. At least 200 BrdU-positive nuclei (S-phase) were counted for each probe. The signal patterns were classified into SS, SD, or DD. On the haploid segment of HL-60 (probes 2∼6), two signal patterns, singlet (S) and doublet (D), were observed. Replication timing of the human 5q23/31, estimated from the FISH analyses, is consistent with that of the cell cycle fractionation studies (see text for details). E, E/M M/L and L stand for early-, early/mid-, mid/late- or late-replicating, respectively.</p

Homologous recombination contributes to the repair of acetaldehyde-induced DNA damage

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1−/− and TDP2−/− cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1−/− and RAD54−/− suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde.</p