Direct-repeat analysis of chromatid interactions during intrachromosomal recombination in mouse cells.

Homologous intrachromosomal recombination between linked genes can involve interactions that are either intramolecular (intrachromatid) or intermolecular (sister chromatid). To assess the relative proportions of chromatid interactions, we report studies of intrachromosomal recombination in mouse L cells containing herpes simplex virus thymidine kinase genes in two alternative configurations of direct repeats. By comparing products of reciprocal exchanges between these two configurations, we conclude that the majority of interactions that give rise to crossover products involve unequally paired sister chromatids after DNA replication. Analyses of an additional class of crossover products that involve discontinuous associated gene conversion suggest that these recombination events involve a heteroduplex DNA intermediate.     

Gene repeat expansion and contraction by spontaneous intrachromosomal homologous recombination in mammalian cells

Homologous recombination (HR) is important in repairing errors of replication and other forms of DNA damage. In mammalian cells, potential templates include the homologous chromosome, and after DNA replication, the sister chromatid. Previous work has shown that the mammalian recombination machinery is organized to suppress interchromosomal recombination while preserving intrachromosomal HR. In the present study, we investigated spontaneous intrachromosomal HR in mouse hybridoma cell lines in which variously numbered tandem repeats of the µ heavy chain constant (Cµ) region reside at the haploid, chromosomal immunoglobulin µ heavy chain locus. This organization provides the opportunity to investigate recombination between homologous gene repeats in a well-defined chromosomal locus under conditions in which recombinants are conveniently recovered. This system revealed several features about the mammalian intrachromosomal HR process: (i) the frequency of HR was high (recombinants represented as much as several percent of the total of recombinants and non-recombinants); (ii) the recombination process appeared to be predominantly non-reciprocal, consistent with the possibility of gene conversion; (iii) putative gene conversion tracts were long (up to 13.4 kb); (iv) the recombination process occurred with precision, initiating and terminating within regions of shared homology. The results are discussed with respect to mammalian intrachromosomal HR involving interactions both within and between sister chromatids.     

Stimulation of intrachromosomal homologous recombination in mammalian cells by an inhibitor of poly(ADP-ribosylation).

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose) polymerase (E.C. 2.4.2.30), on intrachromosomal homologous recombination in mouse Ltk- cells. We used a cell line that contained in its genome two defective Herpes thymidine kinase (tk) genes as closely linked direct repeats. Intrachromosomal homologous recombination events were monitored by selecting for tk-positive segregants that arose during propagation of the cells and recombination rates were determined by fluctuation analysis. We found that growth of cells in the continuous presence of 2mM 3-MB increased intrachromosomal recombination between 3 and 4-fold. Growth of cells in the presence of 2mM m-anisic acid, a non-inhibitory analog of 3-MB, had no effect on intrachromosomal recombination rates. Additionally, we found that 3-MB increased both gene conversions and crossovers to similar extents, adding to the evidence that these two types of intrachromosomal rearrangements share a common pathway. These findings contrast with our previous studies [Waldman, B.C. and Waldman, A.S. (1990) Nucleic Acids Res., 18, 5981-5988] in which we determined that 3-MB inhibits illegitimate recombination and has no effect on extrachromosomal homologous recombination in mouse Ltk- cells. An hypothesis is offered that explains the influence of 3-MB on different recombination pathways in mammalian cells in terms of the role that poly(ADP-ribosylation) plays in DNA break-repair.     

Molecular analysis of sister chromatid recombination in mammalian cells

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of double-strand breaks arising during replication and is thought to be important for the prevention of genomic instability and cancer. Analysis of sister chromatid recombination at a molecular level has been limited by the difficulty of selecting specifically for these events. To overcome this, we have developed a novel "nested intron" reporter that allows the positive selection in mammalian cells of "long tract" gene conversion events arising between sister chromatids. We show that these events arise spontaneously in cycling cells and are strongly induced by a site-specific double-strand break (DSB) caused by the restriction endonuclease, I-SceI. Notably, some I-SceI-induced sister chromatid recombination events entailed multiple rounds of gene amplification within the reporter, with the generation of a concatemer of amplified gene segments. Thus, there is an intimate relationship between sister chromatid recombination control and certain types of gene amplification. Dysregulated sister chromatid recombination may contribute to cancer progression, in part, by promoting gene amplification.     

Mechanisms of nonhomologous recombination in mammalian cells.

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.     

Genetic control of intrachromosomal recombination

Intrachromosomal recombination between direct repeats can occur either as gene conversion events, which maintain exactly the number of repeat units, or as deletions, which reduce the number of repeat units. Gene conversions are classical recombination events that utilize the standard chromosome recombination machinery. Spontaneous deletions between direct repeats are generally recA-independent in E. coli and RAD52-independent in S. cerevisiae. This independence from the major recombination genes does not mean that deletions form through a nonrecombinational process. Deletions have been suggested to result from sister chromatid exchange at the replication fork in a recA-independent process. The same type of exchange is proposed to be RAD52-independent in Saccharomyces cerevisiae. RAD52-dependent events encompass all events that involve the initial steps of a recombination reaction, which include strand invasion to form a heteroduplex intermediate.     

Size-dependent palindrome-induced intrachromosomal recombination in yeast

Palindromic and quasi-palindromic sequences are important DNA motifs found in various cis-acting genetic elements, but are also known to provoke different types of genetic alterations. The instability of such motifs is clearly size-related and depends on their potential to adopt secondary structures known as hairpins and cruciforms. Here we studied the influence of palindrome size on recombination between two directly repeated copies of the yeast CYC1 gene leading to the loss of the intervening sequence (“pop-out” recombination). We show that palindromes inserted either within one copy or between the two copies of the CYC1 gene become recombinogenic only when they attain a certain critical size and we estimate this critical size to be about 70 bp. With the longest palindrome used in this study (150 bp) we observed a more than 20-fold increase in the pop-out recombination. In the sae2/com1 mutant the palindrome-stimulated recombination was completely abolished. Suppression of palindrome recombinogenicity may be crucial for the maintenance of genetic stability in organisms containing a significant number of large palindromes in their genomes, like humans.     

Some genetic properties of intrachromosomal recombination

Summary Intrachromosomal recombination was estimated by the occurrence of unequal crossing over without marker gene exchange in three different, independent tandem duplicaltions in Drosophila melanogaster. Each tandem duplication gave rise to intrachromosomal recombinants. The frequency of intrachromosomal recombination is independent of the genetic length of the tandem duplication. Further, intrachromosomal recombinants were recovered as frequently in ring as in rod X chromosomes implying that the recombination event is not equatable to a single interchromosomal crossover.Communicated by Ch. Auerbach     

Centromere mitotic recombination in mammalian cells

Centromeres are special structures of eukaryotic chromosomes that hold sister chromatid together and ensure proper chromosome segregation during cell division. Centromeres consist of repeated sequences, which have hindered the study of centromere mitotic recombination and its consequences for centromeric function. We use a chromosome orientation fluorescence in situ hybridization technique to visualize and quantify recombination events at mouse centromeres. We show that centromere mitotic recombination occurs in normal cells to a higher frequency than telomere recombination and to a much higher frequency than chromosome-arm recombination. Furthermore, we show that centromere mitotic recombination is increased in cells lacking the Dnmt3a and Dnmt3b DNA methyltransferases, suggesting that the epigenetic state of centromeric heterochromatin controls recombination events at these regions. Increased centromere recombination in Dnmt3a,3b-deficient cells is accompanied by changes in the length of centromere repeats, suggesting that prevention of illicit centromere recombination is important to maintain centromere integrity in the mouse.     

Interchromosomal recombination is suppressed in mammalian somatic cells.

Homologous recombination occurs intrachromosomally as well as interchromosomally, both in mitotic (somatic) cells as well as meiotically in the germline. These different processes can serve very different purposes in maintaining the integrity of the organism and in enhancing diversity in the species. As shown here, comparison of the frequencies of intra- and interchromosomal recombination in meiotic and mitotic cells of both mouse and yeast argues that interchromosomal recombination is particularly low in mitotic cells of metazoan organisms. This result in turn suggests that the recombination machinery of metazoa might be organized to avoid the deleterious effects of homozygotization in somatic cells while still deriving the benefits of species diversification and of DNA repair.     

Telomere recombination in normal mammalian cells

Two mechanisms of telomere length maintenance are known to date. The first includes the use of a special enzymatic telomerase complex to solve the problems that arise during the replication of linear DNA in a normal diploid and part of tumor cells. Alternative lengthening of telomeres (ALT), which is based on the homologous recombination of telomere DNA, represents the second mechanism. Until recently, ALT was assumed to be expressed only in 15–20% of tumors lacking active telomerase and,, together with telomerase reactivation represented one of two possibilities to overcome the replicative senescence observed in somatic mammalian cells due to aging or during cell culturing in vitro. Previously described sporadic cases of combinations of the two mechanisms of telomere length maintenance in several cell lines in vitro were attributed to the experimental design rather than to a real biological phenomenon, since active cellular division without active telomerase was considered to be the “gold standard” of ALT. The present review describes the morphological and functional reorganizations of mammalian telomeres observed with ALT activation, as well as recently observed and well-documented cases of combinations between ALT-like and telomerase-dependent mechanisms in mammalian cells. The possible role of telomere recombination in telomerase-dependent cells is discussed.     

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells.

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage.     

Induction of intrachromosomal homologous recombination in whole plants

The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced severalfold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed.     

Targeted recombination with single-stranded DNA vectors in mammalian cells.

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.     

Thymidylate stress induces homologous recombination activity in mammalian cells.

We studied whether homologous recombination activity in mammalian cells could be induced by thymidylate stress (thymidylate deprivation). In vitro recombination activity in cell extracts was measured with pSV2neo-derived plasmids. When prior to the preparation of extracts, mouse FM3A cells were grown in 5-fluorodeoxyuridine (FdUrd), an inducer of thymidylate stress, the homologous recombination activity was significantly induced, as judged from an increase in the number of neomycin-resistant bacterial colonies. Maximum induction was observed in cells treated with 1 microM FUdR for 16 h. However, 3-8 h of treatment of FM3A cells with the drug followed by an additional 8-16-h incubation in its absence was sufficient to induce the recombination activity while slightly reducing their growth rates. These results indicate that thymidylate stress induces homologous recombination activity in mammalian cells as observed in Escherichia coli and in yeast.     

DNA homologous recombination repair in mammalian cells

DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.     

The same mammalian replicon yields distinct recombination products in different cell lines.

We have observed previously that some chimeric replicons inclusive of a partly duplicated polyomavirus (Py) genome yield unit-length Py DNA (P155) at high frequency when transfected into normal or Py-transformed mouse cells. We demonstrate here that one such replicon generates either P155 or illegitimate recombination products in other mouse cells, transformed by simian virus 40. Use of the polymerase chain reaction indicates that each of the illegitimate products carried a different deletion, but that all deletions mapped within a rather well defined portion of the precursor replicon. Thus, these products were organized as if two hotspots for recombination existed in the Py late-coding region, one being located within or near one of the duplicated sequences characteristic of the chimeric replicon. Since this particular hotspot has already been shown to be involved in the generation of P155, the data reported here could indicate that a single recombination mechanism can yield either homologous (P155) or illegitimate products. How the DNA interacts with certain proteins, such as papovavirus large tumor antigen, could explain why one or the other type of product is formed.