In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein.

The interaction between the RecBCD enzyme of Escherichia coli and the lambda Gam protein was investigated. Two types of experiments were done. In one type, Gam protein was produced by transient induction of the cells lysogenic for lambda cI857gam+. The presence of Gam protein, which inhibits RecBCD nuclease, enabled these cells to support the growth of a gene 2 mutant of bacteriophage T4 (T4 2). The lysogens overproducing the RecB subunit of RecBCD enzyme could titrate Gam protein and thus prevent the growth of T4 2. In contrast, the lysogens overproducing either RecC or RecD retained their capacity for growth of T4 2. It is therefore concluded that the RecB subunit is capable of binding Gam protein. In the second type of experiments, Gam protein was provided by derepressing the gamS gene on the plasmid pSF117 (S. A. Friedman and J. B. Hays, Gene 43:255-263, 1986). The presence of this protein did not interfere with the growth of wild-type cells (which were F-). Gam protein had a certain effect on recF mutants, whose doubling time became significantly longer. This suggests that the recF gene product plays an important role in maintenance of viability of the Gam-expressing cells. Gam protein exerted the most striking effect on growth of Hfr bacteria. In its presence, Hfr bacteria grew extremely slowly, but their ability to transfer DNA to recipient cells was not affected. We showed that the effect on growth of Hfr resulted from the interaction between the RecBCD-Gam complex and the integrated F plasmid.     

Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking activity of RecBCD and together with lambda bet, promotes Chi-independent recombination

Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli to promote phage growth and recombination. Overproduction of the RecC subunit in vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization, revealing that RecC is the target for Abc2 in vivo. UV-induced ATP crosslinking experiments revealed that Abc2 protein does not interfere with the binding of ATP to either the RecB or RecD subunits in the absence of DNA, though it partially inhibits RecBCD ATPase activity. Productive growth of phage P22 in wild-type Salmonella typhimurium correlates with the presence of Abc2, but is independent of the absolute level of ATP-dependent nuclease activity, suggesting a qualitative change in the nature of Abc2-modified RecBCD nuclease activity relative to the native enzyme. In lambda phage crosses, Abc2-modified RecBCD could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam could not. In exonuclease assays designed to examine the polarity of digestion, Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA exonuclease by increasing the rate of digestion of the 5' strand. In this respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered the recombination hotspot Chi. However, unlike Chi-modified RecBCD, Abc2-modified RecBCD still possesses 3' exonuclease activity. These results are discussed in terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22 phage recombination pathway. This mechanism of P22-mediated recombination distinguishes it from phage lambda recombination, in which the phage recombination system (Red) and its anti-RecBCD function (Gam) work independently.     

What makes the bacteriophage λ Red system useful for genetic engineering: molecular mechanism and biological function

Recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. The system, called Red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a 'hyper-rec' state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.     

Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme.

The lambda Gam protein was isolated from cells containing a Gam-producing plasmid. The purified Gam protein was found to bind to RecBCD without displacing any of its subunits. Gam was shown to inhibit all known enzymatic activities of RecBCD: ATP-dependent single- and double-stranded DNA exonucleases, ATP-independent single-stranded endonuclease, and the ATP-dependent helicase. When produced in vivo, Gam inhibited chi-activated recombination in lambda red gam crosses but had little effect on the host's ability to act as a recipient in conjugational recombination. These experiments suggest that RecBCD possesses an additional "unknown" activity that is resistant to or induced by Gam. Additionally, the expression of Gam in recD mutants sensitizes the host to UV irradiation, indicating that Gam alters one or more of the in vivo activities of RecBC(D-).     

The lambda Gam protein inhibits RecBCD binding to dsDNA ends

Inactivation of the Escherichia coli RecBCD enzyme by the lambda Gam protein is an essential step that accompanies the lambda Red proteins for gene replacement using recombineering technology. It has been shown that Gam inhibits all the activities of RecBCD to the same extent. Nonetheless, some in vivo properties of recBCD mutants cannot be mimicked effectively by the expression of gam in vivo. An examination of the mechanism of Gam's inhibition of RecBCD was performed, and it was found that Gam inhibits the binding of RecBCD to double-stranded DNA ends, even if RecBCD is bound to DNA before its interaction with Gam. When ATP is added to the reaction to induce helicase activity, most of the reaction is inhibited by Gam, but residual amounts of unwinding are detected, despite a 40-fold excess of Gam/RecBCD. The same inhibitory effect of Gam was seen on RecBCD that had been modified by the P22 anti-RecBCD protein Abc2, though the inhibitory effect was diminished due to the tighter binding of Abc2-modified RecBCD to double-stranded DNA ends. These data suggest that cells containing Gam-expressing plasmids retain a small amount of uninhibited enzyme. Given the suspected instability of Gam in vivo, care must be taken when interpreting results from experiments containing Gam-inhibited RecBCD species. A revised model is proposed for Gam-induced radioresistance of E. coli to ionizing radiation.     

Interaction of lambda Gam protein with the RecD subunit of RecBCD enzyme increases radioresistance of the wild-type Escherichia coli.

By making use of the gam(+)-plasmid, the so-called gam-dependent radioresistance was studied. This resistance is the result of the interaction between Gam protein (encoded by the gam gene of lambda) and RecBCD enzyme of Escherichia coli. gam-dependent radioresistance is observed in recB+ recC+ recD+ but not in recB+ recC+ recD- cells. It is suggested that Gam protein interacts specifically with the RecD subunit of RecBCD enzyme; the RecBC complex probably retains its activity in the presence of this viral protein.     

Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions.

Plasmids that express the bacteriophage lambda gam gene or the P22 abc2 gene (with and without abc1) at controllable levels were placed in Escherichia coli and tested for effects on the activity of RecBCD. Like Gam, Abc2 inhibited the ATP-dependent exonuclease activity of RecBCD, apparently not by binding to DNA. However, Abc2-mediated inhibition was partial, while Gam-mediated inhibition was complete. Both Abc2 and Gam inhibited host system-mediated homologous recombination in a Chi-containing interval in the chromosome of a hybrid lambda phage; Abc2 inhibited it more strongly than Gam. Gam but not Abc2 spared a phage T4 gene 2 mutant from restriction by RecBCD; Abc2 exhibited weak sparing activity in combination with Abc1 and substantial activity in combination with both Abc1 and P22 homologous recombination function Erf. Either Gam or the combination of the lambda recombination functions Exo and Bet was sufficient to induce a mode of plasmid replication that produced linear multimers. The combination of Abc2, Abc1, and Erf also exhibited this activity. However, Erf was inactive, both by itself and in combination with Abc1; Abc2 had weak activity. These results indicate that Gam and Abc2 modulate the activity of RecBCD in significantly different ways. In comparison with lambda Gam, P22 Abc2 has a weak effect on RecBCD nuclease activity but a strong effect on its recombination-promoting activity.     

Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism.

Two novel types of alleviation of DNA restriction by the EcoKI restriction endonuclease are described. The first type depends on the presence of the gam gene product (Gam protein) of bacteriophage lambda. The efficiency of plating of unmodified phage lambda is greatly increased when the restricting Escherichia coli K-12 host carries a gam+ plasmid. The effect is particularly striking in wild-type strains and, to a lesser extent, in the presence of sbcC and recA mutations. In all cases, Gam-dependent alleviation of restriction requires active recBCD genes of the host and recombination (red) genes of the infecting phage. The enhanced capacity of Gam-expressing cells to repair DNA strand breaks might account for this phenomenon. The second type is caused by the presence of a plasmid in a restricting host lacking RecBCD enzyme. Commonly used plasmids such as the cloning vector pACYC184 can produce such an effect in strains carrying recB single mutations or in recBC sbcBC strains. Plasmid-mediated restriction alleviation in recBC sbcBC strains is independent of the host RecF, RecJ, and RecA proteins and phage recombination functions. The presence of plasmids can also relieve restriction in recD strains. This effect depends, however, on the RecA function in the host. The molecular mechanism of the plasmid-mediated restriction alleviation remains unclear.     

The crystal structure of lambda-Gam protein suggests a model for RecBCD inhibition

In Escherichia coli, RecBCD processes double-stranded DNA breaks during the initial stages of homologous recombination. RecBCD contains helicase and nuclease activities, and unwinds and digests the blunt-ended DNA until a specific eight-nucleotide sequence, Chi, is encountered. Chi modulates the nuclease activity of RecBCD and results in a resected DNA end, which is a substrate for RecA during subsequent steps in recombination. RecBCD also acts as a defence mechanism against bacteriophage infection by digesting linear viral DNA present during virus replication or resulting from the action of restriction endonucleases. To avoid this fate, bacteriophage lambda encodes the gene Gam whose product is an inhibitor of RecBCD. Gam has been shown to bind to RecBCD and inhibit its helicase and nuclease activities. We show that Gam inhibits RecBCD by preventing it from binding DNA. We have solved the crystal structure of Gam from two different crystal forms. Using the published crystal structure of RecBCD in complex with DNA we suggest models for the molecular mechanism of Gam-mediated inhibition of RecBCD. We also propose that Gam could be a mimetic of single-stranded, and perhaps also double-stranded, DNA.     

Chi hotspot activity in Escherichia coli without RecBCD exonuclease activity: implications for the mechanism of recombination

The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCD enzyme, a complex nuclease and DNA helicase regulated by Chi sites (5'-GCTGGTGG-3'). During its unwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions, depending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching of nucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recC mutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but loss of detectable nucleolytic degradation as judged by the growth of mutant T4 and lambda phages and by assay of cell-free extracts. We conclude that RecBCD enzyme's nucleolytic degradation of DNA is not necessary for intracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. We discuss the bearing of these results on current models of RecBCD pathway recombination.     

Gamma-irradiated RecD overproducers become permanent recB-/C- phenocopies for extrachromosomal DNA processing due to prolonged titration of RecBCD enzyme on damaged Escherichia coli chromosome

The RecBCD enzyme of Escherichia coli consists of three subunits RecB, RecC and RecD. RecBCD enzyme activities are regulated by its interaction with recombination hotspot Chi. Biochemical and genetic evidence suggest that interaction with Chi affects RecD subunit, and that RecD polypeptide overproduction antagonizes this interaction, suggesting that intact RecD replaces a Chi-modified one. We used bacteria with fragmented chromosomes due to double-strand breaks inflicted by UV and gamma-irradiation to explore in which way increased concentrations of RecBCD's individual subunits affect DNA metabolism. We confirmed that RecD overproduction alters RecBCD-dependent DNA repair and degradation in E. coli. Also, we found that RecB and RecC overproduction did not affect these processes. To determine the basis for the effects of RecD polypeptide overproduction, we monitored activities of RecBCD enzyme on gamma-damaged chromosomal DNA and, in parallel, on lambda and T4 2 phage DNA duplexes provided at intervals. We found that gamma-irradiated wild-type bacteria became transient, and RecD overproducers permanent recB(-)/C(-) phenocopies for processing phage DNA that is provided in parallel. Since this inability of irradiated bacteria to process extrachromosomal DNA substrates coincided in both cases with ongoing degradation of chromosomal DNA, which lasted much longer in RecD overproducers, we were led to conclude that the RecB(-)/C(-) phenotype is acquired as a consequence of RecBCD enzyme titration on damaged chromosomal DNA. This conclusion was corroborated by our observation that no inhibition of RecBCD activity occurs in gamma-irradiated RecBCD overproducers. Together, these results strongly indicate that RecD overproduction prevents dissociation of RecBCD enzyme from DNA substrate and thus increases its processivity.     

Effect of a null mutation in the priA gene on radioresistance of Escherichia coli

According to Kogoma's model of DNA recombination by replication, the PriA protein is involved in the RecBCD pathway of double-strand break (DSB) repair, which is associated with extensive DNA degradation, at the stage of primosome assembly in D-loops (intermediates of strand exchange at the ends of DSB) for the subsequent switch to DSB-induced DNA resynthesis. Comparable data on possible involvement of the PriA protein in the repair of gamma-ray-induced lethal lesions in cells of the wild-type strain of Escherichia coli (strain AB1157) and in two radiation-resistant mutants Gamr445 and Gamr444 were obtained. In all the three strains examined, the null priA2::kan mutation in the structural priA gene was shown to markedly enhance the radiation sensitivity, causing a two- to threefold increase in the slopes of linear dose-survival curves. In the AB1157 strain, the inactivation of PriA is manifested most clearly in the range of low doses (up to 0.15 kGy) when the priA2::kan mutation had only a slight effect on the radiation resistance of Gamr mutants. It can be assumed that, in these mutants with a decreased level of postradiation DNA degradation, the PriA-dependent RecBCD pathway of DSB repair associated with extensive DNA resynthesis is not essential for the repair of lethal lesions at low doses. However, this pathway becomes crucial at higher doses (> 0.5 kGy) even for radiation-resistant strains, especially for the most resistant Gamr444 mutant.     

Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways

Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in Escherichia coli and constitute the major pathway of error-free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via non-homologous end-joining (NHEJ) catalysed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways.     

Lethality of rep recB and rep recC double mutants of Escherichia coli

A rep mutation in combination with a recB or a recC mutation renders Escherichia coli non-viable. This conclusion is based on the following lines of evidence: (i) double mutants cannot be constructed by P1 transduction; (ii) induction of the λ Gam protein, which inactivates most of the RecBCD activities, is lethal in rep mutants; (iii) rep recBts recCts mutants are not viable at high temperature. The reasons for a requirement for the RecBCD enzyme in rep strains were investigated. Initiation of chromosome replication, elongation and chromosomal segregation do not seem impaired in the rep recBts recCts mutant at the non-permissive temperature. The viability of other rep derivatives was tested. rep recA recD triple mutants are not viable, whereas rep recD and rep recA double mutants are. Inactivation of both exoV activity and recBC -dependent homologous recombination is therefore responsible for the non-viability of rep recBC strains. However, sbcA and sbcB mutations, which render recBC mutants recombination proficient, do not restore viability of rep recBC mutants, indicating that recombination via the RecF or the RecE pathways cannot functionally replace RecBCD-mediated recombination. The specific requirement for RecBCD suggests the occurrence of double-strand DNA breaks in rep strains. Additional arguments in favour of the presence of DNA lesions in rep mutants are as follows: (i) expression of SOS repair functions delays lethality of rep derivatives after inactivation of RecBCD; (ii) sensitivity of rep strains to ultraviolet light is increased by partial inactivation of RecBCD. A model for the recovery of cells from double-strand breaks in rep mutants is discussed.     

A novel cell-free protein synthesis system

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30–45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.     

Effects of recJ, recQ, and recFOR mutations on recombination in nuclease-deficient recB recD double mutants of Escherichia coli

The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5'-->3' exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5'-->3' exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5'-->3' exonuclease is an element of the RecF recombination machinery.     

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1drd-19.

Plasmid R1drd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1drd-19 cells. This paper further investigates the suppressive effect of plasmid R1drd-19 on the recB mutation of E. coli. The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1drd-19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of super oligomerization of the tester pACYC184 plasmid in recB/R1drd-19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by lambda Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. coli chromosomal fragment carrying recBrecCrecD genes and the EcoRI-C fragment of R1drd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.     

Werner syndrome protein associates with gamma H2AX in a manner that depends upon Nbs1

The WRN protein is mutated in the chromosomally unstable Werner syndrome (WS) and the Nbs1 protein is mutated in Nijmegen breakage syndrome (NBS). The Nbs1 protein is an integral component of the M/R/N complex. Although WRN is known to interact with this complex in response to gamma-irradiation, the mechanism of action is unclear. Here, we show that WRN co-localizes and associates with gamma H2AX, a marker protein of DNA double strand breaks (DSBs), after cellular exposure to gamma-irradiation. While the DNA damage-inducible Nbs1 foci formation is normal in WS cells, WRN focus formation is defective in NBS cells. Consistent with this, gamma H2AX colocalizes with Nbs1 in WS cells but not with WRN in NBS cells. The defective WRN-gamma H2AX association in NBS cells can be complemented with wild-type Nbs1, but not with an Nbs1 S343A point mutant that lacks an ATM phosphorylation site. WRN associates with H2AX in a manner dependent upon the M/R/N complex. Our results suggest a novel pathway in which Nbs1 may recruit WRN to the site of DNA DSBs in an ATM-dependent manner.     

Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity.

Escherichia coli strains bearing plasmids expressing phage P22 anti-RecBCD functions abc1 and abc2 were tested for the presence of recBC-like phenotypes. Abc2 induces moderate sensitivity to UV light in wild-type and recD mutant strains but severely sensitizes both recF and recJ mutants. Abc1 has little effect on UV sensitivity in wild-type or recF or recJ mutant hosts but increases the sensitivity of recD mutants to a UV dose of 20 J/m2 about 10-fold. Abc2 induces E. coli to segregate inviable cells during growth, interferes with the growth of lambda red gam chi+ and chi 0 phage (the effect is greater with chi+ phage), inhibits Chi and Chi-like activity as measured by lambda red gam crosses, and prevents SOS induction in response to nalidixic acid; Abc1 has no effect in these tests. Abc2, alone or with Abc1, does not allow the growth of lambda red gam in the presence of a P2 prophage but does not kill the P2 lysogenic host (as lambda Gam does). Finally, Abc2 inhibits conjugational recombination in wild-type cells to the level seen in recBC mutants. These data suggest that Abc2 inhibits the recombination-promoting ability of RecBCD but leaves the exonuclease functions intact.     

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.