WRN RQC

Ken Kitano and colleagues have co-crystallised the RecQ C-terminal (RQC) domain of human WRN bound to a DNA duplex. In their recent Structure paper, they describe how the RQC domain interacts with a blunt end of the duplex and, surprisingly, unpairs a Watson-Crick base pair in the absence of an ATPase domain. The β wing, an extended hairpin motif characteristic of winged-helix motifs, is used as a “separating knife” to wedge between the first and second base pair. However, the recognition helix, a component of helix-turn-helix motifs embedded within DNA grooves, is excluded from the interaction.

These results present a function of the winged-helix motif central to the helicase reaction, and add further paradigmatic insights on the structural biology of RecQ helicases.

doi:10.1016/j.str.2009.12.011

Structural Basis for DNA Strand Separation by the Unconventional Winged-Helix Domain of RecQ Helicase WRN

by

Ken Kitano,  Sun-Young Kim, and Toshio Hakoshima

Volume 18, Issue 2, 10 February 2010, Pages 177-187

introduction of a normal human chromosome 8 corrects Werner Syndrome cells

Kentaro Ariyoshi and colleagues successfully introduced a normal human chromosome 8 into Werner Syndrome (WS) cells(fibroblasts) immortalized by expressing a human telomere reverse transcriptase subunit (hTERT) gene. In their study (published in the Journal of Radiation Research), they demonstrated that the abnormal WS cellular phenotypes like sensitivity to 4-nitroquinoline-1-oxide (4NQO) and hydroxyurea (HU), and chromosomal radiosensitivity at G(2) phase can be corrected by expression of the WRN gene upon introduction of a chromosome 8 via microcell fusion. Their results provide more evidence that the multiple abnormal WS phenotypes (clinical, cellular, and chromosomal) are derived from a primary, but not secondary, defect in the WRN gene.

WRN protects against cytotoxicity of topotecan but not etoposide

Markus Christmann and colleagues recently reported their findings on whether DNA helicases, like WRN, are involved in the repair of topoisomerase inhibitor-induced DNA damage.

“[24] M. Poot, K.A. Gollahon and P.S. Rabinovitch, Werner syndrome lymphoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase, Hum. Genet. 104 (1999), pp. 10–14. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (97)Although the interplay between topo I and WRN has been reported and some early publications are available on WRN and resistance against topo II poisons, there is no systematic analysis of the role of WRN in the sensitivity against topo I and topo II poisons in human cancer cells.”

“… we utilized human U2-OS osteosarcoma cells stably transfected with siRNA specific for the wrn gene. Using this isogenic cell system, we studied for the first time comparatively the role of WRN in cellular resistance to topo I and topo II inhibitors and its involvement in the repair or processing of topo I inhibitor-induced DNA damage.”

Using three different methods (WST assay, colony forming assay, and quantification of apoptosis), they showed that the WRN helicase protects against the cytotoxic effects of the topo I inhibitor topotecan (TPT) but not the topo II inhibitor etoposide (ETO).

Fig. 4. Formation and repair of SSBs and DSBs following TPT treatment. (A) To analyze the TPT-induced formation and repair of DNA strand breaks, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT (left panel) or etoposide (right panel). At the indicated time points, the formation of DNA single-strand breaks was detected via alkaline SCGE. OTM, olive tail moment. Data of the mean ± S.D. of three independent experiments are shown. (B) Determination of DSBs by neutral SCGE in wrn-wt and wrn-kd cells at various times after exposure to 1 μg/ml TPT. Data of at least three independent experiments are shown. (C) To determine the amount of H2AX phosphorylation, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT. At different times after exposure, cells were harvested, protein extracts were prepared and 25 μg were subjected to Western blot analysis. The membrane was incubated with γH2AX specific antibodies. For loading control, ERK2 was detected. To quantify the amount H2AX phosphorylation, the intensity of the strongest band was set to 100%. (D) To determine the formation of γH2AX foci, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT. After the indicated time points, cells were fixed, and the γH2AX foci were visualized using γH2AX specific antibodies using fluorescence microscopy. For each time point, foci in 40 cells were counted and the results of three independent experiments ± S.D. is shown (left panel). A representative experiment is provided in the right panel.

WRN mini-review

In a recent issue of DNA Repair [7 (2008) 1776–1786], Julia Sidorova reviews the role of WRN in preserving DNA integrity during replication and propose that WRN can function in coordinating replication fork progression with replication stress-induced fork remodeling. She further discusses damage tolerance pathways, redundancy, and cooperation with other RecQ helicases.

Fig. 3. Possible scenarios of WRN function in coordinating fork progression with damage repair via control over daughter/daughter duplex expansion and/or half-life. (A) An unproductive daughter/daughter duplex with the 3′ overhang is unwound to redirect damage bypass towards translesion synthesis (TLS). (B) An extension of a daughter/daughter duplex leads to exposure of ssDNA regions of mother strands (for simplicity, only one of the strands is shown coated with RPA). Accumulation of RPA stimulates helicase activity of WRN to limit propagation of daughter/daughter duplex and restore an original fork conformation. (C) Lagging strand synthesis in the presence of a daughter/daughter duplex can lead to formation of long flaps. WRN can prevent their formation by limiting half-life of a daughter/daughter duplex, or stimulate FEN-1 to cleave such flaps once they are formed.