Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

Katsura, Yukitaka; Sasaki, Shigeru; Sato, Masanori; Yamaoka, Kiyoshi; Suzukawa, Kazumi; Nagasawa, Toshiro; Yokota, Jun

doi:10.1016/j.dnarep.2006.12.002

Cited by 28 publications

(26 citation statements)

References 23 publications

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“…In the first group (n = 8), the host sequence directly adjoined the viral sequence with no homology, suggesting a role for nonhomologous end joining (NHEJ) in the integration process (Table 2). In the second group (n = 7), regions of microhomology (1-10 bp) were seen between the host and viral sequences, suggesting microhomology-mediated NHEJ (6,27,28), whereas in the third group (n = 9), stretches of unrecognized ''orphan'' DNA (1-36 bp) were present between the host and viral sequences.…”

Section: Viral Breakpoints and Host-viral Junctional Sequencementioning

confidence: 93%

Characterization of Naturally Occurring HPV16 Integration Sites Isolated from Cervical Keratinocytes under Noncompetitive Conditions

et al. 2008

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As the high-risk human papillomavirus (HPV) integrants seen in anogenital carcinomas represent the end-point of a clonal selection process, we used the W12 model to study the naturally occurring integration events that exist in HPV16-infected cervical keratinocytes before integrant selection. We performed limiting dilution cloning to identify integrants present in cells that also maintain episomes. Such integrants arise in a natural context and exist in a noncompetitive environment, as they are transcriptionally repressed by episome-derived E2. We found that integration can occur at any time during episome maintenance, providing biological support for epidemiologic observations that persistent HPV infection is a major risk factor in cervical carcinogenesis. Of 24 different integration sites isolated from a single nonclonal population of W12, 12 (50%) occurred within chromosome bands containing a common fragile site (CFS), similar to observations for selected integrants in vivo. This suggests that such regions represent relatively accessible sites for insertion of foreign DNA, rather than conferring a selective advantage when disrupted. Interestingly, however, integrants and CFSs did not accurately colocalize. We further observed that local DNA rearrangements occur frequently and rapidly after the integration event. The majority of integrants were in chromosome bands containing a cancer-associated coding gene or microRNA, indicating that integration occurs commonly in these regions, regardless of selective pressure. The cancer-associated genes were generally a considerable distance from the integration site, and there was no evidence for altered expression of nine strong candidate genes. These latter observations do not support an important role for HPV16 integration in causing insertional mutagenesis. [Cancer Res 2008;68(20):8249-59]

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Section: Viral Breakpoints and Host-viral Junctional Sequencementioning

confidence: 93%

Characterization of Naturally Occurring HPV16 Integration Sites Isolated from Cervical Keratinocytes under Noncompetitive Conditions

et al. 2008

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“…Although further analyses may be required, DNA-PKcs does not appear to be required for preferential rAAV integration at DNA palindromes. Since we often found microhomology up to 8 bp at rAAV integration sites in both DNA-PKcs-proficient and -deficient mice, a DNA-PK-independent microhomologydependent alternative end-joining pathway(s) (13,26) might be operative in rAAV integration.…”

Section: Resultsmentioning

confidence: 99%

DNA Palindromes with a Modest Arm Length of ≳20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice

Inagaki

Lewis

et al. 2007

J Virol

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However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of տ20 bp (total length, տ40 bp) are a significant target for rAAV integration. Up to ϳ30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of տ20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

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“…We explored the effect of the proteasome inhibition on NHEJ using an episomal plasmid assay (39). Figure 1A illustrates a schematic cartoon of this assay.…”

Section: Resultsmentioning

confidence: 99%

Inhibitors of the Proteasome Suppress Homologous DNA Recombination in Mammalian Cells

Murakawa¹,

Sonoda²,

Barber

et al. 2007

Cancer Research

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Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA doublestrand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, ;H2AX, or Mre11. These data show that the proteasomemediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways. [Cancer Res 2007;67(18):8536-43]

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Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

Cited by 28 publications

References 23 publications

Characterization of Naturally Occurring HPV16 Integration Sites Isolated from Cervical Keratinocytes under Noncompetitive Conditions

Characterization of Naturally Occurring HPV16 Integration Sites Isolated from Cervical Keratinocytes under Noncompetitive Conditions

DNA Palindromes with a Modest Arm Length of ≳20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice

Inhibitors of the Proteasome Suppress Homologous DNA Recombination in Mammalian Cells

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