After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

Rapp, Alexander; Greulich, Karl Otto

doi:10.1242/jcs.01355

Cited by 105 publications

(85 citation statements)

References 39 publications

(46 reference statements)

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confidence: 56%

“…The density and intensity of ␥-H2AX signals 5 h post-UV irradiation was similarly maintained in both wild-type and udu tu24 embryos (Figure 5,E and F). It has been reported that repair of DNA DSBs takes a longer time compared with DNA SSBs (Rapp and Greulich, 2004). Indeed, our experimental results showed that cells with ␥-H2AX signals were significantly reduced in the trunk and tail regions of both wild-type and udu tu24 embryos after 24 h (data not shown) and further reduced after 31 h ( Figure 5, G and H) UV irradiation.…”

Section: Dna Repair For Dsb Seems Functional In Udu Tu24mentioning

confidence: 52%

“…Comet tail was detected in p53-MO-injected udu tu24 mutants, in which apoptosis is inhibited, excluding the possibility that DNA defect originates from apoptosis-induced DNA fragmentation (Figure 4, F and G). Wild-type embryos subjected to UV treatment, which also causes DSB via propagation of clustered single-stranded breaks (SSBs) that occur at closely spaced DNA lesions (Jenner et al, 2001;Rapp and Greulich, 2004;Garinis et al, 2005), were used as a positive control ( Figure 4H). …”

Section: Loss Of Udu Function Results In Cell Cycle Defectsmentioning

confidence: 99%

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Udu Deficiency Activates DNA Damage Checkpoint

Lim

Chong

Jiang

2009

MBoC

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Udu has been shown to play an essential role during blood cell development; however, its roles in other cellular processes remain largely unexplored. In addition, ugly duckling (udu) mutants exhibited somite and myotome boundary defects. Our fluorescence-activated cell sorting analysis also showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased DNA damage in udu tu24 mutants. We further showed that the extensive p53-dependent apoptosis in udu tu24 mutants is a consequence of activation in the Atm-Chk2 pathway. Udu seems not to be required for DNA repair, because both wild-type and udu embryos similarly respond to and recover from UV treatment. Yeast two-hybrid and coimmunoprecipitation data demonstrated that PAH-L repeats and SANT-L domain of Udu interacts with MCM3 and MCM4. Furthermore, Udu is colocalized with 5-bromo-2 -deoxyuridine and heterochromatin during DNA replication, suggesting a role in maintaining genome integrity. INTRODUCTIONugly duckling (udu tu24 ) mutant was first isolated from the 1996 Tü bingen screen and has been shown to exhibit a short body-axis with massive cell death (Hammerschmidt et al., 1996). Another udu allele, udu sq1 , was isolated in a genetic screen aiming at mutants with defects in hematopoiesis (Liu et al., 2007). Positional cloning revealed that Udu protein encodes a novel nuclear factor consisting of three conserved regions (CR-1, CR-2, and CR-3) that do not share similarity with any known domains, two paired amphipathic ␣-helix like (PAH-L) repeats, and one putative SW13, ADA2, N-Cor and TFIIIB like (SANT-L) domain. The C-terminal PAH-L and SANT-L domains have been shown to be essential in primitive erythroid cell development (Liu et al., 2007). The PAH domain, first identified in yeast SIN3 (Wang et al., 1990), has been demonstrated to mediate protein-protein interactions (Spronk et al., 2000). SIN3 has no intrinsic DNA binding ability and has to be targeted to gene promoters by interacting with DNA binding proteins, thereafter positively and negatively regulating genes involved in diverse cellular functions (Silverstein and Ekwall, 2005). The SANT domain is a highly conserved motif with similarity to Myb DNA binding domain (Aasland et al., 1996), which has been shown to be critical in regulating chromatin accessibility (Boyer et al., 2002(Boyer et al., , 2004.The DNA damage response pathway is a cellular surveillance system that senses the presence of damaged DNA and elicits checkpoint activation and subsequent lesion repair in preventing amplification or loss of genes or chromosomes (Zhou and Elledge, 2000). The critical components of DNA damage checkpoint are two phosphatidyl inositol 3-kinaselike kinase family proteins: Ataxia telangiectasia mutated (ATM) and ATM-and Rad3-related (ATR) (Abraham, 2003;Shiloh, 2003). ATR functions as a sensor and transducer in response to UV light and presumably to genotoxic agents that give rise to stalled replication forks, and subsequently activates Checkp...

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supporting

confidence: 56%

Section: Dna Repair For Dsb Seems Functional In Udu Tu24mentioning

confidence: 52%

Section: Loss Of Udu Function Results In Cell Cycle Defectsmentioning

confidence: 99%

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Udu Deficiency Activates DNA Damage Checkpoint

Lim

Chong

Jiang

2009

MBoC

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“…We next performed immunofluorescence staining for g-H2AX. Since it is suggested that also during S-phase ds breaks occur that are highlighted by g-H2AX staining (Thompson and Limoli, 2003), HaCaT cells were enriched in the G 1 phase of the cell cycle (Supplementary Figures 3A and B) before being irradiated with 60 J/ cm 2 UVA and incubated for 30 min to allow maximal foci induction (Rapp and Greulich, 2004). Furthermore, it was recently shown that UVC radiation induced g-H2AX phosphorylation mainly as a diffuse, even pannuclear staining which was discussed as being dependent on nucleotide excision repair (Marti et al, 2006).…”

Section: Uva Induces Double Strand Breaks In Hacat Keratinocytesmentioning

confidence: 99%

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

et al. 2008

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The role of UVA-radiation-the major fraction in sunlight-in human skin carcinogenesis is still elusive. We here report that different UVA exposure regime (4 Â 5 J/cm 2 per week or 1 Â 20 J/cm 2 per week) caused tumorigenic conversion (tumors in nude mice) of the HaCaT skin keratinocytes. While tumorigenicity was not associated with general telomere shortening, we found new chromosomal changes characteristic for each recultivated tumor. Since this suggested a nontelomere-dependent relationship between UVA irradiation and chromosomal aberrations, we investigated for alternate mechanisms of UVA-dependent genomic instability. Using the alkaline and neutral comet assay as well as c-H2AX foci formation on irradiated HaCaT cells (20-60 J/cm 2 ), we show a dosedependent and long lasting induction of DNA single and double (ds) strand breaks. Extending this to normal human skin keratinocytes, we demonstrate a comparable damage response and, additionally, a significant induction and maintenance of micronuclei (MN) with more acentric fragments (indicative of ds breaks) than entire chromosomes particularly 5 days post irradiation. Thus, physiologically relevant UVA doses cause long-lasting DNA strand breaks, a prerequisite for chromosomal aberration that most likely contribute to tumorigenic conversion of the HaCaT cells. Since normal keratinocytes responded similarly, UVA may likewise contribute to the complex karyotype characteristic for human skin carcinomas.

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“…The exact interaction between HR and NHEJ during DSB repair is not fully understood. Recent studies have shown that both NHEJ and HR factors are recruited to the sites of DSBs (22,23). The NHEJ factors are recruited first, and Rad51 is recruited relatively late (within 1 h after DSB induction).…”

Section: Discussionmentioning

confidence: 99%

TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination

Mao

Seluanov

Jiang

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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TRF2 (telomeric repeat binding factor 2) is an essential component of the telomeric cap, where it forms and stabilizes the T-loop junctions. TRF2 forms the T-loops by stimulating strand invasion of the 3 overhang into duplex DNA. TRF2 also has been shown to localize to nontelomeric DNA double-strand breaks , but its functional role in DNA repair has not been examined. Here, we present evidence that TRF2 is involved in homologous recombination (HR) repair of nontelomeric double-strand breaks. Depletion of TRF2 strongly inhibited HR and delayed the formation of Rad51 foci after ␥-irradiation, whereas overexpression of TRF2 stimulated HR. Depletion of TRF2 had no effect on nonhomologous end-joining, and overexpression of TRF2 inhibited nonhomologous end-joining. We propose, based on our results and on the ability of TRF2 to mediate strand invasion, that TRF2 plays an essential role in HR by facilitating the formation of early recombination intermediates.DNA repair ͉ telomeres T RF2 (telomeric repeat binding factor 2) was first identified as a major telomere binding protein that shields chromosome ends from degradation and from end-to-end fusions (1-3). Mammalian telomeres form large duplex loops in which the single-stranded 3Ј end is embedded within the double-stranded DNA (4). TRF2 generates T-loop structures and recently was shown to bind to DNA junctions (4, 5). The mechanism by which TRF2 facilitates folding of telomeric DNA into T-loops involves binding of TRF2 complexes to DNA and untwisting the neighboring DNA, thereby favoring strand invasion (6).In addition to evidence of TRF2's role in telomere maintenance, there are multiple pieces of evidence that implicate TRF2 in double-strand break (DSB) repair of nontelomeric DNA (7-9). TRF2 localizes to DSB sites at the early stages of cellular response to DSBs, appearing in the first few seconds after DSB induction and leaving as DSBs are being processed (7). TRF2 is phosphorylated by ATM in response to DNA damage (8). The phosphorylated form of TRF2 is not bound to telomeric DNA and is localized to DNA damage sites (8). In turn, overexpression of TRF2 inhibits autophosphorylation of ATM and induction of DNA damage response (9).DSBs are repaired by two major pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ). During HR, the missing information is copied into the DSB site from a homologous sequence, whereas NHEJ joins the broken ends without homology. Several proteins involved in DSB repair interact with TRF2, such as the MRE11/Rad50/NBS1 complex, Ku70, WRN, and BLM (10-12). Thus, TRF2 is phosphorylated in response to DNA damage, localizes to DNA breaks, and interacts with DSB repair proteins, but its functional role in DSB repair is unknown. Here, we examined the role of TRF2 in DSB repair by HR and NHEJ. ResultsReporter Cell Lines for Detection of NHEJ and HR. To study the role of TRF2 in NHEJ and HR, we used an in vivo assay that measures the repair of an induced chromosomal break. The reporter cassette for detection of NHEJ previously ...

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After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

Cited by 105 publications

References 39 publications

Udu Deficiency Activates DNA Damage Checkpoint

Udu Deficiency Activates DNA Damage Checkpoint

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination

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