Article

Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context

Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2011; 286(49):42470-82. DOI: 10.1074/jbc.M111.309252
Source: PubMed

ABSTRACT

During repair of multiple chromosomal double strand breaks (DSBs), matching the correct DSB ends is essential to limit rearrangements. To investigate the maintenance of correct end use, we examined repair of two tandem noncohesive DSBs generated by endonuclease I-SceI and the 3' nonprocessive exonuclease Trex2, which can be expressed as an I-SceI-Trex2 fusion. We examined end joining (EJ) repair that maintains correct ends (proximal-EJ) versus using incorrect ends (distal-EJ), which provides a relative measure of incorrect end use (distal end use). Previous studies showed that ATM is important to limit distal end use. Here we show that DNA-PKcs kinase activity and RAD50 are also important to limit distal end use, but that H2AX is dispensable. In contrast, we find that ATM, DNA-PKcs, and RAD50 have distinct effects on repair events requiring end processing. Furthermore, we developed reporters to examine the effects of the transcription context on DSB repair, using an inducible promoter. We find that a DSB downstream from an active promoter shows a higher frequency of distal end use, and a greater reliance on ATM for limiting incorrect end use. Conversely, DSB transcription context does not affect end processing during EJ, the frequency of homology-directed repair, or the role of RAD50 and DNA-PKcs in limiting distal end use. We suggest that RAD50, DNA-PKcs kinase activity, and transcription context are each important to limit incorrect end use during EJ repair of multiple DSBs, but that these factors and conditions have distinct roles during repair events requiring end processing.

Full-text preview

Available from: jbc.org
  • Source
    • "Spontaneous rat mammary tumors that overexpressed HSF1 that developed in response to DMBA showed aggressive carcinogenesis with inhibition of binding activity between Ku70 and Ku80, suggesting a physiological role for HSF1 in tumor development by inhibition of NHEJ repair activity. Posttranslational modifications of HSF1 such as phosphorylation, acetylation or sumoylation importantly affect the HSF1 activity252627. However, in the case ofwere performed, and each immunoprecipitation reaction mixture was either mock treated (−) or treated with lambda phosphatase (+) (left). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel role for HSF1 as an inhibitor of non-homologous end joining (NHEJ) repair activity was identified. HSF1 interacted directly with both of the N-terminal sequences of the Ku70 and Ku86 proteins, which inhibited the endogenous heterodimeric interaction between Ku70 and Ku86. The blocking of the Ku70 and Ku86 interaction by HSF1 induced defective NHEJ repair activity and ultimately activated genomic instability after ionizing radiation (IR), which was similar to effects seen in Ku70 or Ku80 knockout cells. The binding activity between HSF1 and Ku70 or Ku86 was dependent on DNA damage response such as IR exposure, but not on the heat shock mediated transcriptional activation of HSF1. Moreover, the posttranslational modification such as phosphorylation, acetylation and sumoylation of HSF1 did not alter the binding activities of HSF1-Ku70 or HSF1-Ku86. Furthermore, the defect in DNA repair activity by HSF1 was observed regardless of p53 status. Rat mammary tumors derived using dimethylbenz(a)anthracence revealed that high levels of HSF1 expression which correlate with aggressive malignancy, interfered with the binding of Ku70-Ku80. This data suggests that HSF1 interacts with both Ku70 and Ku86 to induce defective NHEJ repair activity and genomic instability, which in turn suggests a novel mechanism of HSF1-mediated cellular carcinogenesis.
    Preview · Article · Sep 2015 · Oncotarget
  • Source
    • "U2OS cells harboring the DR-GFP, EJ5-GFP and SA-GFP reporters and the H2ax−/− mouse embryonic stem cells were previously described (26–28). The siRNAs used (Fisher/Thermoscientific, sequences as provided by the manufacturer) were siRNF168 (D-007152-18, 5′ ′GAGUAUCACUUACGCGCUA), siBRCA1 (D-003461-06 5′GGGAUACCAUGCAACAUAA or -07 5′GAAGGAGCUUUCAUCAUUC), siRNF8 (J- 006900-05 5′AGAAUGAGCUCCAAUGUAU), siFANCD2 (pool of 4 siRNAs: D-016376-01, -02, -04, -18), si53BP1 (pool of 4 siRNAs: D-003548-01, -02, -04, -05) and non-targeting siCTRL (D-001810-01). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.
    Full-text · Article · May 2014 · Nucleic Acids Research
  • Source
    • "Cells deal with double-stranded DNA breaks through several DNA repair pathways, including HR, EJ, and SSA mediated DNA repair [34], [35]. Reporter cell lines have been established to monitor each of these DNA repair pathways [31]. These cells lines each contain a reporter with a GFP expression cassette disrupted by recognition site(s) for the endonuclease I-SceI. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.
    Full-text · Article · Apr 2014 · PLoS ONE
Show more