INTRODUCTION

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The cDNA generated by reverse transcription during retrotransposon and retrovirus replication can enter the genome by two pathways: it can integrate by using the element-encoded integrase or it can recombine with preexisting elements by using the recombination system of the host (6, 20, 32, 45). Entry into the genome, regardless of the mechanism, alters the host's genetic material. This can have immediate negative consequences, for example, by generating deleterious mutations. Over evolutionary time, however, some retroelement-induced mutations have likely benefited the host by contributing to the genetic variability that is acted upon by natural selection. In addition, there is increasing evidence that retroelements may contribute to specific cellular processes. The clearest example is the role played by retroelements and reverse transcription in telomere maintenance (24).

The evolution of linear chromosomes has presented a particular difficulty for chromosome replication. Chromosome termini become shorter after each round of DNA replication due to the inability to completely replicate chromosome ends. For most organisms, telomerase extends chromosome ends by using telomeric RNA as a template for reverse transcription (5, 15, 23, 26, 33, 34, 47, 53). A clear link between retrotransposons and telomerases has recently been revealed by the cloning of the telomerase catalytic subunit from Euplotes, yeast, and humans (26, 33, 34). Amino acid sequence analysis of the catalytic subunit indicates that it is related to retrotransposon reverse transcriptases. This suggests that during the evolution of linear chromosomes, a reverse transcriptase may have been borrowed from cellular retrotransposons and used to maintain chromosome ends (11).

In some instances, retrotransposons play a direct role in counterbalancing the telomeric sequence loss that occurs as a consequence of DNA replication. Drosophila melanogaster telomeres, for example, are made up of the non-long terminal repeat retrotransposons HeT-A and TART (24). Telomere extension occurs through preferential integration of these elements onto chromosome ends (3, 46). In addition, an increasing number of retroelements have been identified in the telomeric and subtelomeric regions of other species. These include the SART1 and TRAS1 elements of silkworms, the Zepp elements of Chlorella, and the Ty5 retrotransposons of Saccharomyces (17, 37, 50, 54). The presence of these elements at telomeres suggests that they may contribute to telomere maintenance.

Recombination can also compensate for the telomere shortening that results from DNA replication. Amplification of chromosome end sequences can occur through recombination between telomeric or subtelomeric repeats (27, 29, 39, 41, 51). Recombinational amplification of yeast subtelomeric repeats can overcome telomerase defects and suppress the decreased life span phenotype typically associated with such mutations (30, 31). This amplification requires the host's homologous recombination system, namely, the RAD52 gene product.

Our laboratory works on the Ty5 retrotransposons of Saccharomyces, which integrate preferentially into silent chromatin (54, 55). Silent chromatin encompasses yeast telomeres and the silent mating loci and is important in mediating Ty5's target preference (22, 56). Ty5 can recognize domains of silent chromatin, and a single amino acid change at the border of integrase and reverse transcriptase abolishes target specificity (14). We have previously shown that in addition to integration, Ty5 cDNA recombines at high frequencies with homologous substrates (20). In this study, we demonstrate that Ty5 cDNA also recombines preferentially with substrates located in silent chromatin. The preferential amplification of Ty5 at the telomeres through both integration and recombination demonstrates how retrotransposons can contribute to telomere dynamics.

	MATERIALS AND METHODS

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Strains. Yeast strains used in this study were YPH499 (MATa GAL trp163 ura3-52 leu21 his3200 lys2-801 ade2-101), W303-1A (MATa ade2-1 can1-100 his3-11,15 leu2-3 trp1-1 ura3-1), and their isogenic derivatives. The Escherichia coli strain XL1-blue (Stratagene) was used for recombinant DNA manipulations.

Plasmids. Several plasmids (CEN based) carrying either wild-type or mutant Ty5 elements were used to measure recombination: pNK254 (wild-type Ty5), pNK255 [DD(35)E mutation; in-611], pNK530 [DD(35)E and targeting domain double mutant; in-611,1094], pNK535 (polypurine tract [PPT] mutant), pNK536 (U3-tip mutant), and pNK537 (Ty5-HIS3). For coding-sequence mutations, the number refers to the modified amino acid in Ty5's single open reading frame (e.g., in-611). The strains used to calculate cDNA recombination frequencies included YPH499, rad derivatives of YPH499, and W303-1A strains containing various Ty5 insertions (19, 54, 55).

Strain construction. One-step gene disruption (42) was used to make the rad derivatives of YPH499. pRR46 contains the RAD1 gene with the region from 212 to +3853 replaced by LEU2 (the kind gift of L. Prakash and S. Prakash) (40). This plasmid was digested with BamHI before YPH499 was transformed by the lithium acetate method (2). Leu⁺ transformants were confirmed to be rad1 by testing their UV sensitivity and by Southern blot analysis. pSM21 contains the RAD52 gene with a TRP1 insertion (the kind gift of L. Prakash and S. Prakash). After digestion of pSM21 with BamHI and transformation of YPH499, Trp⁺ transformants were confirmed to be rad52 by Southern blot analysis. The rad1 rad52 strain was constructed by transforming the rad1 strain with BamHI-digested pSM21. Trp⁺ transformants were confirmed to be rad52 by Southern blot analysis.

Recombination assays. In assays used to study the effect of integrase, the PPT, the LTR end sequences (U3 tip), or RAD genes on Ty5 cDNA recombination, the plasmid-borne Ty5 elements served as both the donor and recipient elements. pNK254 (wild-type Ty5), pNK255 (in-611), pNK535 (PPT mutant), and pNK536 (U3-tip mutant) were transformed into YPH499 and its derivative rad strains. Three independent transformants were used for all analyses. Transposition assays were conducted as previously described (20). His⁺ colonies (62 to 300) were used to calculate the proportion of plasmid events in all strain combinations. This was calculated by dividing the number of colonies that did not grow on synthetic complete medium without histidine and with 5-FOA (SC-H-5-FOA) by the number of colonies that grew on SC-H medium (see Fig. 1A). Ten individual plasmid events generated by the Ty5 integrase DD(35)E mutant (in-611) in the wild-type strain were then subjected to Southern blot analysis or plasmid rescue followed by restriction mapping to determine whether they were derived by recombination.

	RESULTS

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Effect of Ty5 integrase and Rad52p on Ty5 cDNA recombination. We previously developed a Ty5 transposition assay in S. cerevisiae which used a functional Ty5 element from Saccharomyces paradoxus (54). This element carries a HIS3 marker gene, which is rendered nonfunctional by the presence of an inactivating intron (his3AI). Transposition events are selected after reconstitution of a functional HIS3 gene by transcription, intron splicing, and reverse transcription of Ty5 mRNA (Fig. 1). In addition to integration, Ty5 cDNA also recombines at high frequency with homologous substrates (20). Two classes of recombination products are recovered: elements that have exchanged markers between the cDNA and the substrate and tandem elements. To determine the relationship of the integration and recombination pathways and to determine the host recombination system(s) involved in Ty5 cDNA recombination, Ty5 integrase and RAD gene mutants were characterized for their effects on Ty5 cDNA entry into the genome.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Recombination assays used in this study (originally described in reference 20). (A) Transcription of a plasmid-borne GAL1-Ty5-his3AI element is induced by growing cells in galactose medium. After transcription and reverse transcription, the Ty5 cDNA with its functional HIS3 gene either integrates into chromosomes or recombines with plasmid substrates. This generates His+ colonies. Integration and recombination events are distinguished by whether the His+ cells can grow on SC-H-5-FOA plates. (B) Transcription and reverse transcription of a chromosomal GAL1-Ty5-neoAI element gives rise to Ty5 cDNA carrying the neo gene. This cDNA can either integrate or recombine with chromosomal or plasmid targets. Plasmid recombinants containing both the neo and bla genes will confer an Ampr Neor phenotype when introduced into bacteria; all plasmids will confer an Ampr phenotype. Recombination frequencies are calculated as the product of the recombinant frequency and the proportion of recombinants due to recombination. The arrowheads inside the elements represent LTRs.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Ty5 mutants used in this study. A full-length Ty5 element is shown, with the arrowheads indicating the LTRs. in-611 has a mutation in the DD(35)E domain of integrase; the conserved aspartic acid and glutamic acid residues that define this domain are boxed. in-1094 has a Ser-to-Leu substitution that abolishes targeted integration to silent chromatin (14). U3-tip denotes the first two nucleotide sequences at the 5' end of the 3' LTR. The nucleotide changes in the PPT and U3-tip mutants are noted. All mutations were generated by site-directed mutagenesis. RB, RNA binding domain; PR, protease; IN, integrase; RT, reverse transcriptase; RH, RNase H.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Structural analysis of recombination events generated by a Ty5 integrase mutant. (A) The overall His+ frequency and the proportion of recombination events are shown for the Ty5 integrase mutant and the wild-type Ty5 element. The data were determined by the assay shown in Fig. 1A. (B) Maps of Ty5 donor element and recombination products. The arrowheads represent LTRs. (C) Southern blot analysis of 10 individual His+ colonies generated by the Ty5 integrase mutant (in-611). The upper arrow indicates the restriction fragment of the donor Ty5 element. For lanes with a single hybridizing restriction fragment, this band represents the his3AI marker that has been converted by marker exchange to a functional HIS3 gene. The second hybridizing restriction fragment in some lanes (lower arrow, labeled "II") is derived from tandem elements.

Integration is impaired by mutations in the PPT or the LTR end sequences. Retroelement mRNA contains a primer binding site adjacent to the 5' LTR where minus-strand cDNA synthesis initiates from a complementary host tRNA. A PPT adjacent to the 3' LTR serves as the priming site for plus-strand DNA synthesis. Reverse transcription proceeds through a series of two-strand transfers, ultimately resulting in a linear retroelement cDNA with two flanking LTRs. Integrase recognizes sequences at the ends of the cDNA (10), and the dinucleotide end sequences (TG at the U3 tip and CA at the U5 tip) are nearly invariant among retroviruses and retrotransposons. For Ty1, mutations in the PPT or the U3 and U5 tips largely abolish integration and result in elevated frequencies of recombination (45).

Ty5 cDNA recombines at higher frequencies with substrates associated with silent chromatin. Although Ty5 cDNA recombines at high frequency with plasmid substrates, we have never observed recombination with native S. cerevisiae Ty5 sequences. This could be due to two reasons: native Ty5 elements are bound in silent chromatin, which may render them inaccessible to the host's recombination system, or native Ty5 sequences may be too degenerate to serve as effective substrates with the S. paradoxus element used in the assays. To distinguish between these possibilities, Ty5 insertions (100% identical to the donor element) were introduced at different chromosomal locations and used as recombination substrates (W9-AI, HMR; W77-AI, telomere; W3-AI, control [Fig. 4A]) (19). The control substrate, W3-AI, is transcriptionally active, whereas the substrates in silent chromatin (W9-AI and W77-AI) are transcriptionally repressed (19). Plasmids carrying donor elements were then introduced into these strains, and transposition assays were conducted. Transposition or recombination events were selected by the reconstitution of the HIS3 marker gene. Chromosomal and plasmid events were distinguished by whether the His⁺ cells could grow on SC-H-5-FOA medium. Products of recombination with chromosomal substrates were then identified by Southern blot analysis (data not shown).

View larger version (52K): [in this window] [in a new window]   FIG. 4.   Ty5 cDNA recombines preferentially with substrates located within silent chromatin. (A) Three strains with Ty5 insertions at different chromosomal locations. These Ty5 insertions contain the his3AI marker to facilitate selection of recombination and integration events. YKR039W is an anonymous open reading frame. Ty5-1 is an endogenous Ty5 insertion with flanking LTRs depicted as boxes with arrowheads. Solo endogenous Ty5 LTRs are shown at HMR. E and I designate the HMR transcriptional silencers; T designates the telomeric TG1-3 repeats; X designates the subtelomeric X repeat. (B) Recombination frequency with chromosomal substrates and a donor element with an integrase mutation. A plasmid with a Ty5 integrase mutant (in-611) was introduced into the above-mentioned strains. Transposition assays were conducted, and the chromosomal and plasmid recombination events were distinguished by whether His+ cells could grow on SC-H-5-FOA plates. (C) Recombination frequency with chromosomal substrates and a wild-type (WT) donor element. int, internal; telo, telomeric.

Preferential recombination of Ty5 cDNA with telomeric substrates requires the Ty5-encoded domain that mediates integration specificity. Mutations in a single amino acid (amino acid 1094) near the boundary of Ty5 integrase and reverse transcriptase abolish preferential integration of Ty5 to regions of silent chromatin (Fig. 2) (14). Although it is not yet known whether this mutation lies within integrase or reverse transcriptase, we have designated this allele as in-1094 because of its integration phenotype. Since this amino acid is essential for integration specificity, it may also contribute to the observed preference of Ty5 cDNA to recombine with substrates in silent chromatin. To test this hypothesis, a Ty5 double mutant (in-611,1094) that contained both the DD(35)E mutation (in-611) and the targeting domain mutation (in-1094) was constructed. Transposition and recombination frequencies were tested for the in-611,1094 mutant, and they did not differ significantly from that of the in-611 single mutant (data not shown). Plasmids carrying the integrase double mutant were transformed into the strains with Ty5 substrates at the telomeres (W2-AI, W77-AI, and W84-AI [Fig. 4A]) or in the internal region on chromosome XI (W3-AI [Fig. 4A]). For comparison, the assays were conducted in parallel with in-611. Note that in contrast to the experiments described in the previous section, two additional telomeric recombination substrates were tested. Transposition assays were conducted, and chromosomal and plasmid His⁺ events were distinguished by whether the His⁺ cells could grow on SC-H-5-FOA plates. Chromosomal events were further analyzed by a PCR assay to determine whether they were the result of gene conversion of the endogenous his3-11,15 locus by the HIS3-containing cDNA, recombination with the chromosomal Ty5 substrates, or integration.

Ty5 cDNA recombines with a telomeric substrate when exposed to mating pheromones. In their native state, the transcription of most Ty5 elements is silenced by telomeric chromatin (19). Since transcription is required for transposition, this would prevent cDNA synthesis and subsequent integration or recombination. Transcriptional activation of elements bound in silent chromatin, however, can be achieved by exposure to mating pheromones, indicating that Ty5 normally transposes during mating (19). Transcription and transposition of a telomeric Ty5 element (W77-AI) was induced by exposure to mating pheromone (-factor) to test whether Ty5 cDNA can recombine with this homologous telomeric substrate during pheromone activation. Thirty His⁺ colonies were randomly picked and subjected to Southern blot analysis with an integrase-specific probe. Recombination events were scored as either marker exchange (the presence of only the original Ty5 band) or tandem elements (the original band plus a 6.5-kb band). Integration events were scored as the original band plus a novel band. Of the 30 events characterized, 13 were due to recombination (eight marker exchanges and five tandem elements) (Fig. 5 and data not shown). Two of the five tandem elements were present in multiple copies (Fig. 5, lane **). This suggests that during mating, when Ty5 transcription and transposition naturally occur, Ty5 cDNA also recombines at high frequencies with telomeric elements.

View larger version (77K): [in this window] [in a new window]   FIG. 5.   Recombination of a telomeric Ty5 induced by mating pheromones. His+ colonies were selected after inducing transcription and transposition of a telomeric element (W77-AI) by exposure to -factor. Results of a representative Southern hybridization analysis with 11 randomly picked His+ colonies are shown. One asterisk indicates marker exchanges, and two asterisks indicate tandem elements; unlabeled lanes indicate integration events. Note that the tandem elements appear to contain multiple Ty5s, based on hybridization intensity.

Ty5 LTR is important for tandem element formation but not for marker exchange. Recombination between telomeric and subtelomeric repeats plays a role in telomere maintenance (30, 31, 39, 51). For the S. cerevisiae Y' elements, recombinational amplification can overcome telomerase defects (30). The enhanced recombination of Ty5 cDNA at the telomeres suggests that it could play a similar role, particularly when sequences are amplified through the formation of tandem or multiple-tandem elements. In a previous study, we found that the Ty5 LTR was critical for generating tandem elements (20). Internal Ty5 sequences, although not good recombination substrates, could facilitate tandem element formation when coupled with a Ty5 LTR. We proposed that tandem elements were formed through recombination between the LTRs of the cDNA and the substrate. In our models, internal homology facilitated base pairing and therefore the formation of tandem products.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   Ty5 LTR is critical for tandem element formation but not for marker exchange. The recombination frequencies for different Ty5 substrates were calculated by the assay shown in Fig. 1B. (A) Internal Ty5 sequences or the LTR were used as recombination substrates; only recombination that generated tandem elements was observed. All Ty5 fragments are drawn to scale with the exception of pNK318. (B) Ty5 3'-end sequences with or without an LTR (arrowheads) were used as recombination substrates; marker exchanges were the major class of products.

LTR sequences critical for tandem element formation. Deletion analysis was used to determine whether specific Ty5 LTR sequences are important in forming tandem elements. LTR deletion constructs were used as recombination substrates in the assay shown in Fig. 1B. When one-fourth of the Ty5 LTR was deleted from either the 5' or 3' end, recombination frequencies dropped only 2- to 3-fold (2.6-fold for pNK355 and 2.0-fold for pNK413) (Fig. 7A). This indicated that neither the 5'- nor the 3'-LTR end sequence (65 and 61 bp) is essential for forming tandem elements. However, when one-half or more of the LTR was deleted, the recombination frequencies dropped markedly (at least 18.8-fold for pNK416). This suggests that either these fragments fall below the minimal length required for recombination or they lack essential features that mediate recombination.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   Deletion analysis determines the Ty5 LTR sequences (arrowheads) important in mediating tandem element formation. The recombination frequencies for different Ty5 substrates were calculated by the assay shown in Fig. 1B. (A) LTR deletion constructs were used as recombination substrates. (B) LTR sequences coupled with the immediately adjacent internal sequences were used as recombination substrates.

Despite its importance in recombination, tandem element formation cannot be explained by crossover events initiated within the Ty5 LTR. The importance of the LTR in mediating tandem element formation suggests that recombination is initiated within LTR sequences (20). To determine the recombination initiation sites, point mutations were introduced into two recombination substrates (pNK418 and pNK419) such that a restriction site was gained (BamHI [Fig. 8A]) or destroyed (HindIII [Fig. 8A]). The lengths of the internal Ty5 sequences of these substrates differed. Recombination frequencies with both substrates were first determined and found to be comparable to those with the nonmutagenized substrates (data not shown). Recombination products were then analyzed by restriction mapping and DNA sequencing to determine the pattern of inheritance of the mutations in the tandem elements. If recombination was initiated within the Ty5 LTR, as proposed by our previous models (20), class I products that have both mutations at their 3' ends (Fig. 8A) would be obtained. Surprisingly, and in contrast to our prediction, most products from pNK418 and all products from pNK419 had both mutations in their 5' ends. Sequence analysis of seven randomly selected products for pNK418 and four for pNK419 revealed that all carried both mutations at their 3' ends as well (Fig. 8B). Therefore, despite its critical role in mediating high-frequency tandem element formation, recombination cannot be explained by simple crossover events between the substrate and a full-length Ty5 cDNA.

View larger version (24K): [in this window] [in a new window]   FIG. 8.   Assay to determine the initiation site for tandem element formation. (A) Two point mutations were created in two Ty5 recombination substrates (pNK418 and pNK419). A single asterisk indicates the site of addition of a BamHI site; a double asterisk indicates the location at which a HindIII site was destroyed. Based on our previous model, recombination between the LTRs (arrowheads), which is facilitated by internal sequence homology, would generate class I products that have both markers in the 3' element (20). (B) The observed recombination products for substrates pNK418 and pNK419 (designated class I, II, and III). The distribution of markers was determined by restriction mapping of the 5' elements and by DNA sequencing of the 3' elements.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

Reverse transcription plays an important role in replenishing sequence loss that occurs at the ends of linear chromosomes after replication: telomerase can template sequences onto chromosome ends by reverse transcription, as can transposition through reverse transcription of the D. melanogaster telomeric HeT-A and TART retrotransposons. Recombination between telomeric and subtelomeric repeats can also amplify chromosome end sequences. The yeast Ty5 retrotransposons use both transposition and cDNA recombination to modify telomeric regions.

Ty5 cDNA recombination requires Rad52p. The integrase DD(35)E domain, which is conserved among all retroviruses and retrotransposons, is the catalytic domain of Ty5 integrase. Chromosomal integration events were abolished when the second conserved aspartic acid of the DD(35)E domain and its flanking threonine residue were mutated. The Ty5 integration pathway was also blocked by mutating LTR end sequences (U3 tip), the sites where Ty5 integrase likely binds, or by mutating the PPT. The PPT serves as the priming site for plus-strand cDNA synthesis, and mutations block the formation of double-stranded cDNA, the normal substrate for integration. For all mutations that blocked the integration pathway, cDNA recombination predominated. It is interesting to note that for the PPT mutant, the frequency of cDNA recombination was similar to that of wild-type elements. This suggests that single-stranded, minus-strand cDNA is sufficient to mediate cDNA recombination (see the discussion below). Our observations are very similar to those made when the Ty1 integration pathway was disrupted by mutating either Ty1 integrase, the cDNA termini, or the PPT (45). Mutation of the DD(35)E domain has also been shown to affect in vivo and in vitro integration of Ty3, human immunodeficiency virus type 1, and avian sarcoma virus (21, 48).

Ty5 cDNA recombination and silent chromatin. We have previously shown that silent chromatin is required for Ty5's integration specificity (56). Here we demonstrate that Ty5 also preferentially recombines with homologous substrates in silent chromatin. To more readily recover recombination events, our initial experiments were conducted with a Ty5 integrase mutant (in-611) as a cDNA donor. For substrates located at the telomeres or the silent mating locus HMR, we observed at least a 3.2-fold enhancement in recombination relative to that of a substrate at an internal chromosomal site. Because our internal substrate is transcriptionally active, it can generate integration events, and therefore our values are likely underestimates. The preferential recombination with substrates in silent chromatin was confirmed with a wild-type Ty5 donor element. In addition, the enhancement did not occur simply because our control was a recombinational cold spot. When transformed into yeast cells, linear DNA containing Ty5-HIS3 recombined at slightly higher frequencies with the internal substrate. The preferential recombination was also not due to the transcriptional silencing of substrate elements in silent chromatin, as Ty5 substrates lacking a promoter were approximately as effective as wild-type elements in forming tandem elements or carrying out marker exchanges.

Mechanisms of tandem element formation. Although the Ty5 LTR is required for the formation of tandem elements, it is not involved in marker exchange. This suggests that the two classes of recombination products arise by different mechanisms. For marker exchange, only homology flanking the marker gene is important, whereas for tandem element formation, LTR sequences are required in addition to internal domain homology between the cDNA and substrate. Deletion analysis indicated that substrates missing one-fourth of the LTR at either the 5' end or the 3' end could still form tandem elements efficiently; the sequences missing in these two LTR fragments, therefore, are not essential for recombination. Further deletion (126 bp of the 251-bp LTR) reduced recombination to background levels. This is probably because the length of the LTR substrate fell below what has previously been observed for the minimal efficient segment for recombination in yeast (89 to 250 bp) (18, 49) and not because an essential LTR feature was lost. In support of this, when an 80-bp LTR substrate was extended by including adjacent internal Ty5 sequences, the frequency of tandem element formation was restored. Since this 80 bp did not include essential Ty5 promoter sequences, basal transcription was not involved in recombination. In summary, it is the LTR sequences that mediate recombination and not transcription or protein factors that bind the Ty5 LTR.

View larger version (14K): [in this window] [in a new window]   FIG. 9.   Models for tandem element formation. The models illustrate the formation of class III recombination products (Fig. 8), although the same models can also generate class I and class II products. Class I and II products are formed, depending on the site of strand invasion (A) or the extent of recession (B). A single asterisk indicates the BamHI mutation; two asterisks indicate the HindIII mutation. Thick arrows represent LTRs; thin dotted lines represent newly synthesized DNA; dashed thick arrows represent recessed LTR sequences. (A) Step 1. The 3' end of partial-minus-strand Ty5 cDNA (single stranded) invades the substrate. Step 2. branch migration and strand displacement continue through the 5' LTR. The Holliday junction is cleaved (small arrowhead). Step 3. The LTR at the 5' end of the invading cDNA displaces the newly synthesized LTR at the 3' end. This forms a looped intermediate. The 3' OH at the site of the Holliday junction is ligated to the 5' end of the cDNA. The strand with the free 5' end, released from Holliday junction cleavage, is trimmed and ligated (small arrowhead). Step 4. The mismatch repair system cleaves the top strand of the mismatched region. DNA repair is carried out, with the bottom strand as a template, followed by ligation. Step 5. Recombination products are formed, with markers in both the 5' and 3' elements. (B) Step 1. The cDNA is recessed beyond the region containing the two markers. Step 2. The 3' end of the cDNA invades the substrate, and DNA synthesis repairs the gap. Step 3. The Holliday structure is resolved; the small arrows indicate the sites of cleavage. Step 4. Recombination products are formed, with markers in both elements.