No abstract

DNA ligase III and the essential protein XRCC1 are present at greatly reduced levels in the xrcc1 mutant CHO cell line EM-C11. Cell-free extracts prepared from these cells were used to examine the role of the XRCC1 gene product in DNA base excision repair in vitro. EM-C11 cell extract was partially defective in ligation of base excision repair patches, in comparison to wild type CHO-9 extracts. Of the two branches of the base excision repair pathway, only the single nucleotide insertion pathway was affected; no ligation defect was observed in the proliferating cell nuclear antigen-dependent pathway. Full complementation of the ligation defect in EM-C11 extracts was achieved by addition to the repair reaction of recombinant human DNA ligase III but not by XRCC1. This is consistent with the notion that XRCC1 acts as an important stabilizing factor of DNA ligase III. These data demonstrate for the first time that xrcc1 mutant cells are partially defective in ligation of base excision repair patches and that the defect is specific to the polymerase ␤-dependent single nucleotide insertion pathway. DNA base excision repair (BER)1 counteracts the mutagenic and cytotoxic effects of various kinds of base alterations that do not significantly distort the secondary structure of the double helix. A common intermediate of this pathway is the abasic (AP) site, that arises as a consequence of removal of altered bases by DNA-N-glycosylases or as spontaneous detachment of normal bases from the deoxyribose-phosphate backbone. It has been calculated that 2000 -10000 AP sites arise each day in a mammalian cell under physiological conditions (1). Therefore, the task of BER is engaging and important, and data obtained in Escherichia coli and transgenic mice show that this process is essential for survival (2-4). We have recently shown that, in addition to the polymerase ␤-dependent single nucleotide insertion pathway previously investigated in mammalian cells (5), a distinct proliferating cell nuclear antigen (PCNA)-dependent pathway is also present that incorporates a repair patch size of 7-14 nucleotides extending 3Ј to the site of the lesion (6). Our knowledge of the enzymology of the two pathways has several gaps. In particular, the enzymology of the ligation step is poorly defined. A role for the XRCC1 protein has been suggested on the basis of the sensitivity of xrcc1 mutant cell lines (the CHO derivatives EM9 and EM-C11) to agents that introduce DNA base damage (7, 8) and because of their reduced rate of single-strand break rejoining following exposure to ionizing radiation or alkylating agents (9, 10). Consistent with a role for XRCC1 in DNA ligation and BER is its observed interaction with DNA ligase III and DNA polymerase ␤ (7, 11, 12). Here, we have examined directly the role of XRCC1 and DNA ligase III in mammalian BER using a cellfree system. We report for the first time that (i) xrcc1 mutant cells are partially defective in ligation of BER patches and (ii) the defect involves only the polymerase ␤-dependent single nucle...

It has been reported that cancer stem cells may contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. We have examined DNA repair in five stem and nonstem glioma cell lines. The population doubling time was significantly increased in stem compared with nonstem cells, and enhanced activation of Chk1 and Chk2 kinases was observed in untreated CD133 + compared with CD133 À cells. Neither DNA base excision or single-strand break repair nor resolution of pH2AX nuclear foci were increased in CD133 + compared with CD133 À cells. We conclude that glioma stem cells display elongated cell cycle and enhanced basal activation of checkpoint proteins that might contribute to their radioresistance, whereas enhanced DNA repair is not a common feature of these cells. (Mol Cancer Res 2009;7(3):383 -92)

Fanconi Anemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration-free induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA-patient bone marrow cells.

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX S136A/S139A ) indicate that phosphorylated H2AX (cH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to cH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.