I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila.

As a step toward the development of a homologous recombination system in Drosophila, we have developed a methodology to target double-strand breaks (DSBs) to a specific position in the Drosophila genome. This method uses the mitochondrial endonuclease I-SceI that recognizes and cuts an 18-bp restriction site. We find that >6% of the progeny derived from males that carry a marker gene bordered by two I-SceI sites and that express I-SceI in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I-SceI sites revealed that in the majority of the cases, the introduction of DSBs at the I-SceI sites resulted in the complete deletion of the marker gene; the other events were associated with partial deletion of the marker gene. We discuss a number of applications for this novel technique, in particular its use to study DSB repair mechanisms.     

Drosophila ATR in double-strand break repair

The ability of a cell to sense and respond to DNA damage is essential for genome stability. An important aspect of the response is arrest of the cell cycle, presumably to allow time for repair. Ataxia telangiectasia mutated (ATM) and ATR are essential for such cell-cycle control, but some observations suggest that they also play a direct role in DNA repair. The Drosophila ortholog of ATR, MEI-41, mediates the DNA damage-dependent G2-M checkpoint. We examined the role of MEI-41 in repair of double-strand breaks (DSBs) induced by P-element excision. We found that mei-41 mutants are defective in completing the later steps of homologous recombination repair, but have no defects in end-joining repair. We hypothesized that these repair defects are the result of loss of checkpoint control. To test this, we genetically reduced mitotic cyclin levels and also examined repair in grp (DmChk1) and lok (DmChk2) mutants. Our results suggest that a significant component of the repair defects is due to loss of MEI-41-dependent cell cycle regulation. However, this does not account for all of the defects we observed. We propose a novel role for MEI-41 in DSB repair, independent of the Chk1/Chk2-mediated checkpoint response.     

Multiple-pathway analysis of double-strand break repair mutations in Drosophila

The analysis of double-strand break (DSB) repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage “decision circuit” in which the DSB is first placed into one of two pools from which a specific pathway is then selected.     

A genetic screen for DNA double-strand break repair mutations in Drosophila

The study of DNA double-strand break (DSB) repair has been greatly facilitated by the use of rare-cutting endonucleases, which induce a break precisely at their cut sites that can be strategically placed in the genome. We previously established such a system in Drosophila and showed that the yeast I-SceI enzyme cuts efficiently in Drosophila cells and those breaks are effectively repaired by conserved mechanisms. In this study, we determined the genetic requirements for the repair of this I-SceI-induced DSB in the germline. We show that Drosophila Rad51 and Rad54 are both required for homologous repair by gene conversion, but are dispensable for single-strand annealing repair. We provided evidence suggesting that Rad51 is more stringently required than Rad54 for intersister gene conversion. We uncovered a significant role of DNA ligase IV in nonhomologous end joining. We conducted a screen for candidate mutations affecting DSB repair and discovered novel mutations in genes that include mutagen sensitive 206, single-strand annealing reducer, and others. In addition, we demonstrated an intricate balance among different repair pathways in which the cell differentially utilizes repair mechanisms in response to both changes in the genomic environment surrounding the break and deficiencies in one or the other repair pathways.     

Differential usage of alternative pathways of double-strand break repair in Drosophila

Double-strand DNA breaks can be repaired by any of several alternative mechanisms that differ greatly in the nature of the final repaired products. We used a reporter construct, designated "Repair reporter 3" (Rr3), to measure the relative usage of these pathways in Drosophila germ cells. The method works by creating a double-strand break at a specific location such that expression of the red fluorescent protein, DsRed, in the next generation can be used to infer the frequency at which each pathway was used. A key feature of this approach is that most data come from phenotypic scoring, thus allowing large sample sizes and considerable precision in measurements. Specifically, we measured the proportion of breaks repaired by (1) conversion repair, (2) nonhomologous end joining (NHEJ), or (3) single-strand annealing (SSA). For conversion repair, the frequency of mitotic crossing over in the germ line indicates the relative prevalence of repair by double Holliday junction (DHJ) formation vs. the synthesis-dependent strand annealing (SDSA) pathway. We used this method to show that breaks occurring early in germ-line development were much more frequently repaired via single-strand annealing and much less likely to be repaired by end joining compared with identical breaks occurring later in development. Conversion repair was relatively rare when breaks were made either very early or very late in development, but was much more frequent in between. Significantly, the changes in relative usage occurred in a compensatory fashion, such that an increase in one pathway was accompanied by decreases in others. This negative correlation is interpreted to mean that the pathways for double-strand break repair compete with each other to handle a given breakage event.     

The effect of gap length on double-strand break repair in Drosophila

When a double-strand break has a gap between the broken ends, the missing information can be restored through synthesis from a homologous template. Here we address the question of how long such a gap can be before this process fails. We measured the frequency of homologous repair in the Drosophila germ line following the creation of gaps of specific sizes ranging from 3.8 to 210 kb. We found that gaps of 11 kb.     

Cernunnos/XLF: a new player in DNA double-strand break repair

Non-homologous end-joining (NHEJ) is the predominant repair pathway for DNA double-strand breaks (DSBs) in vertebrates and also plays a crucial role in V(D)J recombination of immunoglobulin genes. Cernunnos/XLF is a newly identified core factor for NHEJ, and its defect causes a genetic disease characterized by neural disorders, immunodeficiency and increased radiosensitivity. Cernunnos/XLF has at least two distinct functions in NHEJ. Cernunnos/XLF interacts with and stimulates the XRCC4/DNA ligase IV complex, which acts at the final ligation step in NHEJ. In living cells, Cernunnos/XLF quickly responds to DSB induction and accumulates at damaged sites in a Ku-dependent but XRCC4-independent manner. These observations indicate that Cernunnos/XLF plays a unique role in bridging damage sensing and DSB rejoining steps of NHEJ. Recent crystallographic analyses of the homodimeric Cernunnos/XLF protein provide structural insights into the Cernunnos/XLF functions. These studies offer important clues toward understanding the molecular mechanism for NHEJ-defective diseases.     

DNA double-strand break repair: a relentless hunt uncovers new prey

A major pathway for repair of DNA double-strand breaks is nonhomologous end-joining (NHEJ). In this issue of Cell, and report the discovery of a new NHEJ factor called Cernunnos-XLF. Both groups report that this protein is mutated in a rare inherited human syndrome characterized by severe immunodeficiency, developmental delay, and hypersensitivity to agents that cause DNA double-strand breaks.     

Temporal analysis of meiotic DNA double-strand break formation and repair in Drosophila females

Using an antibody against the phosphorylated form of His2Av (γ-His2Av), we have described the time course for the series of events leading from the formation of a double-strand break (DSB) to a crossover in Drosophila female meiotic prophase. MEI-P22 is required for DSB formation and localizes to chromosomes prior to γ-His2Av foci. Drosophila females, however, are among the group of organisms where synaptonemal complex (SC) formation is not dependent on DSBs. In the absence of two SC proteins, C(3)G and C(2)M, the number of DSBs in oocytes is significantly reduced. This is consistent with the appearance of SC protein staining prior to γ-His2Av foci. However, SC formation is incomplete or absent in the neighboring nurse cells, and γ-His2Av foci appear with the same kinetics as in oocytes and do not depend on SC proteins. Thus, competence for DSB formation in nurse cells occurs with a specific timing that is independent of the SC, whereas in the oocytes, some SC proteins may have a regulatory role to counteract the effects of a negative regulator of DSB formation. The SC is not sufficient for DSB formation, however, since DSBs were absent from the heterochromatin even though SC formation occurs in these regions. All γ-His2Av foci disappear before the end of prophase, presumably as repair is completed and crossovers are formed. However, oocytes in early prophase exhibit a slower response to X-ray–induced DSBs compared to those in the late pachytene stage. Assuming all DSBs appear as γ-His2Av foci, there is at least a 3:1 ratio of noncrossover to crossover products. From a comparison of the frequency of γ-His2Av foci and crossovers, it appears that Drosophila females have only a weak mechanism to ensure a crossover in the presence of a low number of DSBs.     

DNA双链断裂修复的选择性调控机制

在各种DNA损伤中,DNA双链断裂(double-strand break,DSB)是最为严重的一种,快速准确地修复DSB对维持基因组稳定性起着至关重要的作用。真核生物细胞通过一系列复杂的信号转导途径激活对DSB的修复,其中最为重要的是同源重组和非同源末端连接机制。最近的研究表明,这两种方式在DSB修复的早期是相互竞争的关系,其选择在很大程度上受到53BP1及同源蛋白质的调控。将讨论53BP1作为DSB修复途径的核心因子,在染色质水平整合BRCA1、Ct IP等修复因子和多种组蛋白修饰构成的信号途径,介导同源重组和非同源末端连接通路选择的分子机制。     

DNA double-strand break repair and chromosome translocations

Translocations are genetic aberrations that occur when a broken fragment of a chromosome is erroneously rejoined to another chromosome. The initial event in the creation of a translocation is the formation of a DNA double-strand break (DSB), which can be induced both under physiological situations, such as during the development of the immune system, or by exogenous DNA damaging agents. Two major repair pathways exist in cells that repair DSBs as they arise, namely homologous recombination, and non-homologous end-joining. In some situations these pathways can function inappropriately and rejoin ends incorrectly to produce genomic rearrangements, including translocations. Translocations have been implicated in cancer because of their ability to activate oncogenes. Due to selection at the level of the DNA, the cell, and the tissue certain forms of cancer are associated with specific translocations that can be used as a tool for diagnosis and prognosis of these cancers.     

Chromatin dynamics in DNA double-strand break repair

DNA double-strand breaks (DSBs) occur in the context of a highly organized chromatin environment and are, thus, a significant threat to the epigenomic integrity of eukaryotic cells. Changes in break-proximal chromatin structure are thought to be a prerequisite for efficient DNA repair and may help protect the structural integrity of the nucleus. Unlike most bona fide DNA repair factors, chromatin influences the repair process at several levels: the existing chromatin context at the site of damage directly affects the access and kinetics of the repair machinery; DSB induced chromatin modifications influence the choice of repair factors, thereby modulating repair outcome; lastly, DNA damage can have a significant impact on chromatin beyond the site of damage. We will discuss recent findings that highlight both the complexity and importance of dynamic and tightly orchestrated chromatin reorganization to ensure efficient DSB repair and nuclear integrity. This article is part of a Special Issue entitled: Chromatin in time and space.     

Healing the wounds inflicted by sleeping beauty transposition by double-strand break repair in mammalian somatic cells

The Sleeping Beauty (SB) element is a useful tool to probe transposon-host interactions in vertebrates. We investigated requirements of DNA repair factors for SB transposition in mammalian cells. Factors of nonhomologous end joining (NHEJ), including Ku, DNA-PKcs, and Xrcc4 as well as Xrcc3/Rad51C, a complex that functions during homologous recombination, are required for efficient transposition. NHEJ plays a dominant role in repair of transposon excision sites in somatic cells. Artemis is dispensable for transposition, consistent with the lack of a hairpin structure at excision sites. Ku physically interacts with the SB transposase. DNA-PKcs is a limiting factor for transposition and, in addition to repair, has a function in transposition that is independent from its kinase activity. ATM is involved in excision site repair and affects transposition rates. The overlapping but distinct roles of repair factors in transposition and in V(D)J recombination might influence the outcomes of these mechanistically similar processes.     

Genetic analysis of double-strand break repair in Escherichia coli.

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.