FANCD2 influences replication fork processes and genome stability in response to clustered DSBs

<p>Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy.</p

A schematic drawing showing a model of the repair for TMZ and ACNU induced DNA damage.

<p>A schematic drawing showing a model of the repair for TMZ and ACNU induced DNA damage.</p

siRNA silencing of <i>FANCD1/BRCA2</i> in glioblastoma A172 cells after ACNU and TMZ treatments.

<p><b>a</b>, expression analysis of FANCD1/BRCA2 in cells transfected with siRNA for human <i>FANCD1/BRCA2</i> or the negative control siRNA using Western blots. Actin was used as a loading control, and the relative ratios of protein were normalized using actin levels. <b>b</b>, effect of siRNA silencing of <i>FANCD1/BRCA2</i> on cellular sensitivity to ACNU and TMZ. <b>c</b>, effect of siRNA silencing of <i>FANCD1/BRCA2</i> on cell growth after ACNU or TMZ treatment. Open columns, negative control siRNA; closed columns, <i>FANCD1/BRCA2</i> siRNA. Columns show the mean of three independent experiments; the bars indicate the SD. An asterisk (*) indicates significant difference (<i>P</i><0.05). Two asterisks (**), <i>P</i><0.01; three asterisks (***), <i>P</i><0.001.</p

Rad51 foci formation in glioblastoma A172 cells after ACNU and TMZ treatments.

<p><b>a</b>, typical photo. <b>b</b>, open columns, negative control siRNA; closed columns, <i>FANCD1/BRCA2</i> siRNA. Columns show the mean of two independent experiments; bars indicate the SD.</p

Sensitivity to TMZ.

<p><b>a</b>, <i>FANCwt</i> cells (open circles), <i>FANCA^−/−C^−/−</i> cells (closed triangles), <i>FANCC^−/−</i> cells (closed squares), and <i>FANCA^−/−</i> cells (closed circles); <b>b</b>, <i>FANCwt</i> cells (open circles) and <i>FANCD2^−/−</i> cells (closed circles); <b>c</b>, <i>FANCGwt</i> cells (open circles), <i>FANCGrev</i> cells (closed triangles), and <i>FANCG^−/−</i> cells (closed circles); <b>d</b>, <i>FANCD1wt</i> cells (open circles), <i>FANCD1rev</i> cells (closed triangles), and <i>FANCD1mt</i> cells (closed circles). Each point represents the mean of three independent experiments; bars indicate the SD.</p

Relative <i>D₅₀</i> values (% compared to proficient cells) for ACNU (open columns) and TMZ (closed columns).

<p>Two asterisks (**), <i>P</i><0.01; three asterisks (***), <i>P</i><0.001.</p