Repair Responses to DNA Damage: Enzymatic Pathways in E coli and Human Cells

Bacteria and eukaryotic cells employ a variety of enzymatic pathways to remove damage from DNA or to lessen its impact upon cellular functions. Most of these processes were discovered in Escherichia coli and have been most extensively analyzed in this organism because suitable mutants have been isolated and characterized. Analogous pathways have been inferred to exist in mammalian cells from the presence of enzyme activities similar to those known to be involved in repair in bacteria, from the analysis of events in cells treated with DNA damaging agents, and from the analysis of the few naturally occurring mutant cell types. Excision repair of pyrimidine dimers produced by UV in E coli is initiated by an incision event catalyzed by a complex composed of uvrA, uvrB, and uvrC gene products. Multiple exonuclease and polymerase activities are available for the subsequent excision and resynthesis steps. In addition to the constitutive pathway, which produces short patches of 20–30 nucleotides, an inducible excision repair process exists that produces much longer patches. This long patch pathway is controlled by the recA-lexA regulatory circuit and also requires the recF gene. It is apparently not responsible for UV-induced mutagenesis. However, the ability to perform inducible long patch repair correlates with enhanced bacterial survival and with a major component of the Weigle reactivation of bacteriophage with double-strand DNA genomes. Mammalian cells possess an excision repair pathway similar to the constitutive pathway in E coli. Although not as well understood, the incision event is at least as complex, and repair resynthesis produces patches of about the same size as the constitutive short patches. In mammalian cells, no patches comparable in size to those produced by the inducible pathway of E coli are observed. Repair in mammalian cells may be more complicated than in bacteria because of the structure of chromatin, which can affect both the distribution of DNA damage and its accessibility to repair enzymes. A coordinated alteration and reassembly of chromatin at sites of repair may be required. We have observed that the sensitivity of digestion by staphylococcal nuclease (SN) of newly synthesized repair patches resulting from excision of furocoumarin adducts changes with time in the same way as that of patches resulting from excision of pyrimidine dimers. Since furocoumarin adducts are formed only in the SN-sensitive linker DNA between nucleosome cores, this suggests that after repair resynthesis is completed, the nucleosome cores in the region of the repair event do not return exactly to their original positions. We have also studied excision repair of UV and chemical damage in the highly repeated 172 base pair α DNA sequence in African green monkey cells. In UV irradiated cells, the rate and extent of repair resynthesis in this sequence is similar to that in bulk DNA. However, in cells containing furocoumarin adducts, repair resynthesis in α DNA is only about 30% of that in bulk DNA. Since the frequency of adducts does not seem to be reduced in α DNA, it appears that certain adducts in this unique DNA may be less accessible to repair. Endonuclease V of bacteriophage T4 incises DNA at pyrimidine dimers by cleaving first the glycosylic bond between deoxyribose and the 5′ pyrimidine of the dimer and then the phosphodiester bond between the two pyrimidines. We have cloned the gene (denV) that codes for this enzyme and have demonstrated its expression in uvrA recA and uvrB recA cells of E coli. Because T4 endonuclease V can alleviate the excision repair deficiency of xeroderma pigmentosum when added to permeabilized cells or to isolated nuclei after UV irradiation, the cloned denV gene may ultimately be of value for analyzing DNA repair pathways in cultured human cells.     

DNA sequence changes in mutations induced by ultraviolet light in the gpt gene on the chromosome of Escherichia coli uvr+ and urvA cells

Summary Sequence changes in mutations induced by ultraviolet light are reported for the chromosomal Escherichia coli gpt gene in almost isogenic E. coli uvr ⁺ and excision-deficient uvrA cells. Differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr ⁺ cells. This conclusion is confirmed by analysis of published results for genes in both uvr ⁺ and uvr ^– cells, showing a similar selective removal of mutagenic products from the transcribed strand of the E. coli lacI gene and of the lambda phage cl repressor gene. Comparison of these data with published results for ultraviolet mutagenesis of gpt on a chromosome in Chinese hamster ovary cells showed that a mutagenic hot spot in mammalian cells is not present in E. coli; the possibility is suggested that the hot spot might arise from localized lack of excision repair. Otherwise, mutagenesis in hamster cells appeared similar to that in E. coli uvr ⁺ cells, except there appears to be a smaller fraction of single-base additions and deletions (frameshifts) in mammalian than in bacterial cells. Phenotypes of 6-thioguanine-resistant E. coli showed there is a gene (or genes) other than gpt involved in the utilization of thioguanine by bacteria.     

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli’s and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10¹ to 10⁷ copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.     

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated λ red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA⁺ or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD⁺ allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged λ DNA and that its antirecombinogenic activity is operative at elevated levels only.     

Escherichia coli mutY-dependent mismatch repair involves DNA polymerase I and a short repair tract

Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of -[³²P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3 side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.     

Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation

Summary The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied. For this purpose a polylinker sequence flanked by 165 by direct repeats was inserted within the tet gene of pBR327. This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the Kpnl restriction site of the polylinker. Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung ⁺ but not in extracts from ung ^– E. coli strains). Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet ⁺ gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants. Transformation of E. coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue. The frequency of deletions is increased tenfold in an ung ⁺ E. coli strain in comparison with an ung ^– strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.     

Repair of O6-methyl-guanine residues in DNA takes place by a similar mechanism in extracts from hela cells,human liver,and rat liver

Extracts from HeLa S₃ cells, human liver, and rat liver were found to contain an activity that transfers the methyl group from O⁶-methyl-guanine residues in DNA to a cysteine residue of an acceptor protein. The molecular weights of the acceptor proteins in HeLA cells and human liver are 24,000 ± 1,000 and 23,000 ± 1,000. respectively. Assuming that each acceptor molecule is used only once, the average number of acceptor molecules in HeLa cells was calculated to be about 50,000. The extracts also contained 3-methyl-adenine-DNA glycosylase activity and 7-methyl-guanine-DNA glycosylase activity, although the latter activity was not detected in extracts from human liver in our assay system. Thus, the three major alkylation products resulting from the effect of methylating agents, such as N-methyl-N-nitroso urea, can all be repaired in animal cells. Pretreatment of HeLa cells with N-methyl-N′-nitro-N-nitrosoguanidinc (0.1 μg/ml) strongly reduced the capacity of HeLa cell extracts to repair O⁶-methyl-guanine residues, while the activity of three DNA-N-glycosylases was essentially unaltered. This inactivation was not caused by a direct methylation of the enzyme by the carcinogen. The results demonstrate that the mechanism of repair of O⁶-methyl-guaninc residues, in DNA is strikingly similar in E coli and animal cells, including humans.     

大肠杆菌细胞DNA复制、修复和重组途径的衔接

以大肠杆菌为例围绕相关领域的研究动态进行分析和总结.DNA复制、损伤修复和重组过程的相互作用关系研究是当今生命科学研究的前沿和热点之一.越来越多的研究表明,在分子水平上,DNA复制、损伤修复和重组过程既彼此独立,又相互依存.上述途径可以通过许多关键蛋白质之间的相互作用加以协调和整合,并籍此使遗传物质DNA得到有效的维护和忠实的传递.需要指出的是,基于许多细胞内关键蛋白及其功能在生物界中普遍保守性的事实,相信来自大肠杆菌有关DNA复制、修复和重组之间的研究成果也会对相关真核生物的研究提供借鉴.     

Mutagenic DNA repair genes on plasmids from the ''pre-antibiotic era''

Summary Resistance transfer factors are natural conjugative plasmids encoding antibiotic resistance. Some also encode mutagenic DNA repair genes giving resistance to DNA damage and induced mutagenesis. It has been shown that antibiotic resistance has been acquired by recent transposition events; however, we show here that mutagenic repair genes existed much earlier on these types of plasmids. Conjugative plasmids from eight incompatibility groups from the Murray collection of pre-antibiotic era enterobacteria were tested for complementation of mutagenic repair-deficient Escherichia coli umuC36. Although none of these plasmids carry transposon-encoded drug resistance genes, IncI1 and IncB plasmids were identified which restored ultraviolet resistance and induced mutability to umuC36 mutants. Furthermore they increased the UV resistance and induced mutability of wild-type E. coli, Klebsiella aerogenes and Citrobacter intermedius, thus showing that they could confer a general selective advantage to a variety of hosts. Like know mutagenic repair genes, complementation by these plasmid genes required the SOS response of the host cell. Nucleotide hybridisation showed that these plasmids harboured sequences similar to the impCAB locus, the mutagenic repair operon of modern-day IncI1 plasmids. The evolution of mutagenic repair genes is discussed.     

Protective action of melatonin against oxidative DNA damage—Chemical inactivation versus base-excision repair

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 μM and its direct metabolite N¹-acetyl-N²-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 μM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.     

The tail that wags the dog: p12, the smallest subunit of DNA polymerase δ, is degraded by ubiquitin ligases in response to DNA damage and during cell cycle progression

DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication. Human Pol δ is a heterotetramer whose p12 subunit is degraded in response to DNA damage, leading to the in vivo conversion of Pol δ4 to Pol δ3. Two E3 ubiquitin ligases, RNF8 and CRL4^Cdt2, participate in the DNA damage-induced degradation of p12. We discuss how these E3 ligases integrate the formation of Pol δ3 and ubiquitinated PCNA for DNA repair processes. CRL4^Cdt2 partially degrades p12 during normal cell cycle progression, thereby generating Pol δ3 during S phase. This novel finding extends the current view of the role of Pol δ3 in DNA repair and leads to the hypothesis that it participates in DNA replication. The coordinated regulation of licensing factors and Pol δ3 by CRL4^Cdt2 now opens new avenues for control of DNA replication. A parallel study of Pol δ4 and Pol δ3 in Okazaki fragment processing provides evidence for a role of Pol δ3 in DNA replication. We discuss several new perspectives of the role of the 2 forms of Pol δ in DNA replication and repair, as well the significance of the integration of p12 regulation in DNA repair and cell cycle progression.     

High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families

We have examined high affinity interactions of chick brain microtubule proteins with ³⁵S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1–4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65–75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. ³⁵S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM^–1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three ³⁵S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720–1.740 g/cm³; 1.750–1.765 g/cm³). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.     

Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage

Background

Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn²⁺ protection of DNA repair enzymes.

Thymine DNA Glycosylase Is a CRL4Cdt2 Substrate

The E3 ubiquitin ligase CRL4^Cdt2 targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4^Cdt2 while binding to PCNA. In vertebrates, CRL4^Cdt2 promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4^Cdt2 targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4^Cdt2. Our results expand the group of vertebrate CRL4^Cdt2 substrates to include a bona fide DNA repair enzyme.     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

Participation of the intracellular enzymes in the control of mutational processes

Summary The influence of UV-specific endonuclease and medium composition on the frequency and spectrum of genic mutations in Escherichia coli KI2 uvr ⁺ (with normal repair enzymes) and urv A6 (defective in UV-specific endonuclease) was studied. Mutations at the locus glu (gene controlling assimilation of glucose) were induced by ultra-violet irradiation and hydroxylamine treatment. To identify mutant colonies, triphenyl tetrazolium chloride (TTC) was added to the medium since it coloured the mutant colonies bright crimson and readily permitted distinction between pure mutant clones (complete mutations) and mixed clones (mosaic or sector mutations).A maximum mutation frequency after UV-irradiation was observed in E. coli uvr ⁺ cells but not in the E. coli uvr A6 strain. The curve of mutagenesis with a maximum was found in both studied strains after treatment by hydroxylamine which did not cause DNA damage recognized by UV-specific endonuclease.The highest frequency of mutations (at the point of maximum) in the series of experiments with enriched growth medium was almost 10 times higher than in the series of the experiments with poor medium.It was established that in bacteria with normal repair enzymes the frequency of complete mutations was higher than the frequency of mosaic mutations. It was also observed that the rate of UV-mutagenesis was higher in the case of E. coli uvr ⁺.The study of the distribution of mosaic mutant sectors in experiments with bacteria suspended in either a nutrient broth or a buffer during UV-irradiation revealed that the size of mutant sectors was rather variable and that the differences in the number of nucleoids per cell did not always determine the distribution of mutant sector sizes.Abbreviations HA Hydroxylamine hydrochloride - TTC Triphenyl tetrazolium chloride - TCA Trichloroacetic acid Other papers of this series are: Soyfer 1972; Soyfer et al. 1977; Soyfer and Kartel 1978     

NAD+: The convergence of DNA repair and mitophagy

ATM is a 350 kDa serine/threonine kinase best known for its role in DNA repair and multiple cellular homeostasis pathways. Mutation in ATM causes the disease ataxia telangiectasia (A-T) with clinical features including ataxia, severe cerebellar atrophy and Purkinje cell loss. In a cross-species study, using primary rat neurons, the roundworm C. elegans, and a mouse model of A-T, we showed that loss of ATM induces mitochondrial dysfunction and compromised mitophagy due to NAD⁺ insufficiency. Remarkably, NAD⁺ repletion mitigates both the DNA repair defect and mitochondrial dysfunction in ATM-deficient neurons. In C. elegans, NAD⁺ repletion can clear accumulated dysfunctional mitochondria through restoration of compromised mitophagy via upregulation of DCT-1. Thus, NAD⁺ ties together DNA repair and mitophagy in neuroprotection and intimates immediate translational applications for A-T and related neurodegenerative DNA repair-deficient diseases.     

Defective ATM‐Kap‐1‐mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model

ATM‐mediated phosphorylation of KAP‐1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24^?/? mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM‐Kap‐1 signaling is compromised in Zmpste24^?/? MEFs, leading to defective DNA damage‐induced chromatin remodeling. Knocking down Kap‐1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24^?/? MEFs. Thus, ATM‐Kap‐1‐mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.     

Structure and organization of rhodophyte and chromophyte plastid genomes: implications for the ancestry of plastids

Summary Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15–20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms⁺ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.