John H. Wilson

Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction.

Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed.

Manipulating the mammalian genome by homologous recombination.

Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies—chimeric nucleases and triplex-forming oligonucleotides—for stimulating recombination in cells.

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.

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.

Triplex-directed modification of genes and gene activity

Oligonucleotides offer enormous potential for manipulating gene function in cells and, as such, constitute a promising new class of pharmaceutical agents. Oligonucleotides that form triple helices (triplexes) at specific DNA sequences in defined genes can be used to reduce transcription selectively, to introduce site-specific mutations or to stimulate gene-specific targeted recombination.

Transcription promotes contraction of CAG repeat tracts in human cells

Induced transcription through CAG repeats in human cells increases repeat contraction ∼15-fold in both confluent and proliferating cells. Repeats are stabilized against contraction by siRNA knockdown of MSH2, MSH3 or XPA, but not by knockdown of MSH6, XPC or FEN1. These results define a pathway for CAG·CTG repeat contraction that is initiated by transcription, depends on elements of mismatch and nucleotide-excision repair and does not require DNA replication.

R loops stimulate genetic instability of CTG·CAG repeats

Transcription stimulates the genetic instability of trinucleotide repeat sequences. However, the mechanisms leading to transcription-dependent repeat length variation are unclear. We demonstrate, using biochemical and genetic approaches, that the formation of stable RNA·DNA hybrids enhances the instability of CTG·CAG repeat tracts. In vitro transcribed CG-rich repeating sequences, unlike AT-rich repeats and nonrepeating sequences, form stable, ribonuclease A-resistant structures. These RNA·DNA hybrids are eliminated by ribonuclease H treatment. Mutation in the rnhA1 gene that decreases the activity of ribonuclease HI stimulates the instability of CTG·CAG repeats in E. coli. Importantly, the effect of ribonuclease HI depletion on repeat instability requires active transcription. We also showed that transcription-dependent CTG·CAG repeat instability in human cells is stimulated by siRNA knockdown of RNase H1 and H2. In addition, we used bisulfite modification, which detects single-stranded DNA, to demonstrate that the nontemplate DNA strand at transcribed CTG·CAG repeats remains partially single-stranded in human genomic DNA, thus indicating that it is displaced by an RNA·DNA hybrid. These studies demonstrate that persistent hybrids between the nascent RNA transcript and the template DNA strand at CTG·CAG tracts promote instability of DNA trinucleotide repeats.

Role of ERCC1 in removal of long non‐homologous tails during targeted homologous recombination

The XpF/Ercc1 structure‐specific endonuclease performs the 5′ incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/Rad10 endonuclease from Saccharomyces cerevisiae. In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination. To determine whether XpF/Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1+ and ERCC1− cell lines with insertion vectors having long or short terminal non‐homologies flanking each side of a double‐strand break. No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast. However, profound differences were observed in types of APRT+ recombinants recovered from ERCC1− cells using targeting vectors with long terminal non‐homologies—almost complete ablation of gap repair and single‐reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1+ cells. These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non‐homologous tails from invading homologous strands.

Transcription-Induced CAG Repeat Contraction in Human Cells Is Mediated in Part by Transcription-Coupled Nucleotide Excision Repair

ABSTRACT Expansions of CAG repeat tracts in the germ line underlie several neurological diseases. In human patients and mouse models, CAG repeat tracts display an ongoing instability in neurons, which may exacerbate disease symptoms. It is unclear how repeats are destabilized in nondividing cells, but it cannot involve DNA replication. We showed previously that transcription through CAG repeats induces their instability (Y. Lin, V. Dion, and J. H. Wilson, Nat. Struct. Mol. Biol. 13:179-180). Here, we present a genetic analysis of the link between transcription-induced repeat instability and nucleotide excision repair (NER) in human cells. We show that short interfering RNA-mediated knockdown of CSB, a component specifically required for transcription-coupled NER (TC-NER), and knockdowns of ERCC1 and XPG, which incise DNA adjacent to damage, stabilize CAG repeat tracts. These results suggest that TC-NER is involved in the pathway for transcription-induced CAG repeat instability. In contrast, knockdowns of OGG1 and APEX1, key components involved in base excision repair, did not affect repeat instability. In addition, repeats are stabilized by knockdown of transcription factor IIS, consistent with a requirement for RNA polymerase II (RNAPII) to backtrack from a transcription block. Repeats also are stabilized by knockdown of either BRCA1 or BARD1, which together function as an E3 ligase that can ubiquitinate arrested RNAPII. Treatment with the proteasome inhibitor MG132, which stabilizes repeats, confirms proteasome involvement. We integrate these observations into a tentative pathway for transcription-induced CAG repeat instability that can account for the contractions observed here and potentially for the contractions and expansions seen with human diseases.

Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation.

Extra nucleotides (termed filler DNA) are commonly found at the junctions of genetic rearrangements in mammalian cells. The filler DNA at immune system rearrangements, which are called N regions, are generated at VDJ joints primarily by terminal deoxynucleotidyl transferase. However, the origin of filler DNA at genetic rearrangements in nonlymphoid cells is uncertain. In an analysis of more than 200 junctions that arose by circularization of transfected linear DNA (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986), we found 18 junctions with extra nucleotides exactly at the point of circularization. Analysis of these 18 junctions indicated that nonlymphoid cells could add extra nucleotides to the ends of duplex DNA. The characteristics of the extra nucleotides at these junctions and at 31 other rearrangement junctions from nonlymphoid cells were quite similar, suggesting that many genetic rearrangements may pass through a stage with free DNA ends. A comparison of the filler DNA at these 49 nonimmune system rearrangements with 97 N regions derived from immune system rearrangements suggested that lymphoid and nonlymphoid cells use different mechanisms for insertion of filler DNA, as expected from the absence of detectable terminal deoxynucleotidyl transferase in cells from nonlymphoid tissues. The filler DNAs at a smaller group of 22 translocations associated with cancer had features in common with both immune and nonimmune system rearrangements and therefore may represent a mixture of these two processes. Mechanisms that might account for the presence of filler DNA in nonlymphoid cells are discussed.