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.
"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.
Nucleic Acids Research 05/2014; 42(12). DOI:10.1093/nar/gku421 · 9.11 Impact Factor
"Cells deal with double-stranded DNA breaks through several DNA repair pathways, including HR, EJ, and SSA mediated DNA repair , . Reporter cell lines have been established to monitor each of these DNA repair pathways . 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.
PLoS ONE 04/2014; 9(4):e94773. DOI:10.1371/journal.pone.0094773 · 3.23 Impact Factor
"The U2OS cell line containing a stably integrated copy of the direct repeat-green fluorescent protein (DR-GFP) construct was kindly provided by Dr. Maria Jasin, and the frequency of HR was measured according to previous publications , . Briefly, Liopfectamine RNAiMAX (Invitrogen) was used to transfect 75 picomoles of either scramble control siRNA (Santa Cruz sc-37007) or ATR siRNA (Santa Cruz sc-29763) into 1×105 DR-GFP U2OS cells using the reverse transfection method according to the manufacturer’s protocol. "
[Show abstract][Hide abstract] ABSTRACT: DNA replication fork stalling or collapse that arises from endogenous damage poses a serious threat to genome stability, but cells invoke an intricate signaling cascade referred to as the DNA damage response (DDR) to prevent such damage. The gene product ataxia telangiectasia and Rad3-related (ATR) responds primarily to replication stress by regulating cell cycle checkpoint control, yet it's role in DNA repair, particularly homologous recombination (HR), remains unclear. This is of particular interest since HR is one way in which replication restart can occur in the presence of a stalled or collapsed fork. Hypomorphic mutations in human ATR cause the rare autosomal-recessive disease Seckel syndrome, and complete loss of Atr in mice leads to embryonic lethality. We recently adapted the in vivo murine pink-eyed unstable (pun) assay for measuring HR frequency to be able to investigate the role of essential genes on HR using a conditional Cre/loxP system. Our system allows for the unique opportunity to test the effect of ATR loss on HR in somatic cells under physiological conditions. Using this system, we provide evidence that retinal pigment epithelium (RPE) cells lacking ATR have decreased density with abnormal morphology, a decreased frequency of HR and an increased level of chromosomal damage.
PLoS ONE 03/2014; 9(3):e91222. DOI:10.1371/journal.pone.0091222 · 3.23 Impact Factor
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