Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

Abstract

In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of doublestrand break (DSB) repair. Rejoining of DSB produced by decay of 125 I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the 125 I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin 125 I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the 125 I-TFO within its target site. We show that NHEJ of 125 I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after 125 I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.Mammalian genomes constantly suffer a variety of types of damage, of which double-strand breaks (DSB) 1 are considered the most dangerous. DSB may arise spontaneously in the cell or may be induced by exogenous agents, such as ionizing radiation. The estimation that mammalian cells suffer at least 10 spontaneous DSB/day suggests that efficient repair of DSB is critical for cell survival (1). Failure to do so can result in deleterious genomic rearrangements, cell cycle arrest, or cell death.Recent studies have revealed that DSB in the genomes of higher eukaryotes can be repaired by at least three different pathways (2): (i) Homologous recombination repair, the most accurate process, is able to restore the original sequence at the break. Because of its strict dependence on extensive sequence homology, this mechanism is suggested to be active mainly during the S and G 2 phases of the cell cycle (3, 4). (ii) Singlestranded annealing is another homology-dependent but less accurate process that can repair DSB between direct repeats and thereby produces mainly interstitial deletions (4). (iii) Nonhomologous DNA end joining (NHEJ) comprises at least two different processes (5). The major and best investigated NHEJ pathway depends on the Ku70/80 heterodimer, the catalytic subunit of the DNA-dependent protein kinase, DNA ligase IV, and its essential co-factor XRCC4 (6, 7). In contrast to homologous recombination repair and single-stranded annealing, NHEJ can operate in the absence of sequence homology (although short sequence homologies, so-called microhomologies, may facilitate the process) and is able to rejoin broken ends directly (2). This process is supposed to occur mainly in the G 0 and G 1 phases of th...

Intron creation and DNA repair

“…Some NHEJ events, however, involve capture of filler DNA leading to large insertions [64][65][66][67]. The relative frequencies of these events depend on the nature of DNA damage, the taxonomic position of the species concerned, and in experimental settings, the conditions used [64,65,68]. NHEJ-mediated insertion events also vary with respect to junction borders and sequence complexity of the DNA integrated.…”

“…Various experimental studies unearthed a potpourri of DNA sequences as inserts. Integrated sequences include simple repeats, such as (GT) n [64], G(T) 20 [68] or (GAGAA) 9 (AAAGG) 3 (AAGGG) 3 [66], fragments of expression vectors (applied to generate chromosomal DSBs), genomic DNA from bacteria (presumably contaminants of transfection vectors), deliberately added sequences, and DNA of unknown origin [66,67]. Thus, it appears that DNA of any source, when available, can be recruited for NHEJ.…”

Non-Homologous DNA End Joining in Anticancer Therapy

“…Other in vitro NHEJ assays have been used to measure end joining of naturally occurring radiation-induced DSBs [28][29]. The NHEJ repair reactions with all purified proteins Ku, DNA-PK cs , Artemis, DNA ligase IV/XRCC4, Pol µ and Pol λ have been recently reconstituted in vitro [30] thereby opening up a new and exciting era that will allow the testing of drugs that affect NHEJ pathways and are useful in the battle against cancer.…”

Double-strand Break Repair Machinery is Sensitive to UV Radiation

“…[9][10][11][12][13] Some of the deletions would change the frame of the coding sequence and result in a complete change in the type of protein produced. Moreover, large deletions, no matter whether they are in frame or not, would also lead to complete protein inactivation.…”

“…Most studies conducted so far were unable to identify point mutations as outcomes of the end-joining repair. [9][10][11][12][13] Careful analysis of the experimental approaches used in the past led us to the conclusion that in the vast majority of cases, the experimental design limited the possibility of identifying point mutations as outcomes of the strand break repair and were discarded as possible sequencing or PCR mistakes. However, point mutations could frequently arise as a consequence of DNA end processing upon a microhomology search.…”