New insights into the Fanconi anemia pathway from an isogenic FancG hamster CHO mutant

Tebbs, Robert S.; Hinz, John M.; Yamada, N. Alice; Wilson, James B.; Salazar, Edmund P.; Thomas, C.; Jones, Irene M.; Jones, Nigel J.; Thompson, Larry H.

doi:10.1016/j.dnarep.2004.06.013

Cited by 60 publications

(58 citation statements)

References 68 publications

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“…Nevertheless, mild UV sensitivity of FA cells has been reported (30). Our results showing BPDE sensitivity of FANCD2(Ϫ) cells are also consistent with a role for the FA pathway in processing and repair of bulky adducts at stalled replication forks.…”

Section: Discussionsupporting

confidence: 89%

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Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Song

Palle

Gurkar

et al. 2010

Journal of Biological Chemistry

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Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for crosstalk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA monoubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

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Section: Discussionsupporting

confidence: 89%

“…1F). Other workers have also reported mild UV sensitivity of FA cell lines (30), further indicating a role for FANCs in tolerance of bulky DNA lesions. Taken together, our results show that the FA pathway participates in cellular responses to bulky DNA adducts such as BPDE-induced lesions.…”

Section: Fa Pathway Activation In Response To Bpde-induced Bulkymentioning

confidence: 77%

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Song

Palle

Gurkar

et al. 2010

Journal of Biological Chemistry

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“…The FA pathway provides significant protection against the lethal effects of agents that cause replication stalling and FA-defective cells are very sensitive indicators of the presence of a variety of DNA lesions that block advancing replication forks. Some time ago, FANCG-defective hamster cells were reported to be unusually sensitive to 6-TG (20). We confirmed this hypersensitivity in FANCA-deficient MEFs and showed that abrogation of the FA pathway also confers 6-TG sensitivity in human cells.…”

Section: Discussionsupporting

confidence: 77%

“…An early study (20) reported that FANCGdefective Chinese hamster ovary cells are even more sensitive to 6-TG than to acknowledged ICL-inducing agents such as mitomycin C and diepoxybutane. This extreme 6-TG sensitivity was also apparent in FANCA À/À MEFs that were more than four-fold more sensitive to 6-TG than their FANCA þ/þ counterparts (Fig.…”

Section: Mmr-independent 6-tg Sensitivitymentioning

confidence: 99%

Oxidation-Mediated DNA Cross-Linking Contributes to the Toxicity of 6-Thioguanine in Human Cells

Brem

Karran

2012

Cancer Research

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The thiopurines azathioprine and 6-mercaptopurine have been extensively prescribed as immunosuppressant and anticancer agents for several decades. A third member of the thiopurine family, 6-thioguanine (6-TG), has been used less widely. Although known to be partly dependent on DNA mismatch repair (MMR), the cytotoxicity of 6-TG remains incompletely understood. Here, we describe a novel MMR-independent pathway of 6-TG toxicity. Cell killing depended on two properties of 6-TG: its incorporation into DNA and its ability to act as a source of reactive oxygen species (ROS). ROS targeted DNA 6-TG to generate potentially lethal replication-arresting DNA lesions including interstrand cross-links. These triggered processing by the Fanconi anemia and homologous recombination DNA repair pathways. Allopurinol protected against 6-TG toxicity by acting as a ROS scavenger and preventing DNA damage. Together, our findings provide mechanistic evidence to support the proposed use of thiopurines to treat HR-defective tumors and for the coadministration of 6-TG and allopurinol as an immunomodulation strategy in inflammatory disorders. Cancer Res; 72(18); 4787-95. Ó2012 AACR.

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“…CHO cells expressing a dominant-negative form of Rad51 (62) (the kind gift of Dr B. Lopez), Xrcc3-deficient irs1SF cells (63), the FancG knock-out line KO40 (64), and the Brca2-deficient line V-C8 (65) have all been described. V-C8 cells complemented by expression of wild type Brca2 were the generous gift of Drs.…”

Section: Methodsmentioning

confidence: 99%

Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

Liu

Majumdar

Liu

et al. 2010

Journal of Biological Chemistry

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Double strand breaks (DSBs) can be repaired by homology independent nonhomologous end joining (NHEJ) pathways involving proteins such as Ku70/80, DNAPKcs, Xrcc4/Ligase 4, and the Mre11/Rad50/Nbs1 (MRN) complex. DSBs can also be repaired by homology-dependent pathways (HDR), in which the MRN and CtIP nucleases produce single strand ends that engage homologous sequences either by strand invasion or strand annealing. The entry of ends into HDR pathways underlies protocols for genomic manipulation that combine site-specific DSBs with appropriate informational donors. Most strategies utilize long duplex donors that participate by strand invasion. Work in yeast indicates that single strand oligonucleotide (SSO) donors are also active, over considerable distance, via a single strand annealing pathway. We examined the activity of SSO donors in mammalian cells at DSBs induced either by a restriction nuclease or by a targeted interstrand cross-link. SSO donors were effective immediately adjacent to the break, but activity declined sharply beyond ϳ100 nucleotides. Overexpression of the resection nuclease CtIP increased the frequency of SSO-mediated sequence modulation distal to the break site, but had no effect on the activity of an SSO donor adjacent to the break. Genetic and in vivo competition experiments showed that sequence conversion by SSOs in the immediate vicinity of the break was not by strand invasion or strand annealing pathways. Instead these donors competed for ends that would have otherwise entered NHEJ pathways. Non-homologous end joining (NHEJ)3 repair is un-templated, as there is no requirement, or even apparent use, for an informational reference sequence acting in trans. NHEJ might be regarded as a collection of operations (end processing by nucleases, limited single strand exposure of the ends, fill in by polymerases, ligation), without requirement for a specific order of events (1). In mammalian cells the best characterized version of NHEJ, often termed "classical" (C-NHEJ) (2, 3), utilizes several factors, including Ku70/80, Artemis, XLF, DNAPKcs, Xrcc4/DNA ligase 4, pol, pol, and XLF/Cernunnos (4 -8).Although precise joining of restriction enzyme cleaved ends can occur, the pathway is frequently mutagenic, resulting in the introduction of short deletions at the break site (9 -11).Deletions at the DSB are sometimes bounded by short homology elements located on either side of the break. These have been described as the product of a microhomology mediated end joining (MMEJ) pathway (5, 12). Deletions without microhomologies are also recovered and recent work suggests that the MRN complex plays an important role in both versions of deletional NHEJ (2, 13-15). A backup pathway involving poly(ADP-ribose) polymerase 1 and ligase III has been described as well (16,17). It should be noted that whether end joining in the absence of one of the canonical factors is evidence of different discrete pathways, or simply a variation on a fundamental scheme, is a matter for discussion (7). Nonetheless, NHEJ is the dom...

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New insights into the Fanconi anemia pathway from an isogenic FancG hamster CHO mutant

Cited by 60 publications

References 68 publications

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Oxidation-Mediated DNA Cross-Linking Contributes to the Toxicity of 6-Thioguanine in Human Cells

Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

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