An L1 element harbouring the Tx1L EN retrotransposes in an

Each of the two parallel β-sheets of AP-like ENs are formed by one half of the EN peptide chain. Partial EN swaps were therefore reasoned to lead to steric incompatibility at the extensive interface of the β-sheet structures. Consequently, only one block swap experiment was carried out with Tx1L EN: pNZ39 is a construct with almost the complete EN of L1.3 being substituted by the corresponding polypeptide sequence of Tx1L EN (Fig. 17A). In this setting, at least the EN domain should be able to fold correctly. Analogous to pNZ33, the EN substitution was limited to the region between residues 15 and 230 of L1 ORF2p (see 3.3.2.1, Fig. 16).

Tx1L EN (10-230)

pJM101/L1.3 100.0±18.5

pNZ39 0.18±0.09 pNZ49 0.02±0.02 pNZ51 0.27±0.07 pNZ63 0.15±0.08 pNZ64 0.13±0.10 pNZ65 0.13±0.13 pNZ66 0.19±0.12 L1 EN

Reporter % WT activity construct

L1-EN

(1-14) L1-EN

(231-239) D N D H

D N D D H N D H N D

H

pJM101/L1.3 pNZ39 pNZ49 pNZ51

pNZ64 pNZ65 pNZ66

0,0 0,1 0,2 0,3

pNZ63

80 100

pJM101 pNZ49 pNZ51 pNZ63 pNZ64pNZ39 pNZ65 pNZ66

143 145

205 230

B

C

D N D H + D702A mutation in RT

AAA_n

EN RT C oen

D702

A

hyg ori amp EBNA-1 oriP

pJM101/L1.3

A A A

A

A A

A A A

Fig. 17: Reporter construct pNZ39 harbouring an L1/Tx1L EN chimera is retrotransposition-competent but transposes in an apparently EN-independent manner. (A) Schematic representation of the structures of L1/Tx1L EN chimeras. pJM101/L1.3 carrying a wild-type L1 EN (orange) was used as positive control. Tx1L-derived sequences are coloured green. The four catalytical residues D143, N145, D205 and H230 are shown and their replacement by alanines is indicated in red. The names of the reporter constructs and their activity relative to wild-type L1 are indicated on the right. (B) Representative results of retrotransposition assays performed with the indicated reporter constructs are shown. (C) Graphic representation of the results of the retrotransposition assays (n=6). Due to the differences in activity of wild-type and mutant L1s, the y-axis is split into two parts with different scales.

RESULTS 69 In seven independent experiments, this construct reproducibly yielded a very low number

(2-16) of G418 resistant colonies (Fig. 17B,C). This number was always above the levels of the negative control pJM105 and of the R1Bm block swaps (0.18±0.09% wild-type activity of pNZ39 versus 0.03% for pJM105 and ≤0.01% for pNZ33-37). This suggested that the L1 element with a transplanted Tx1L EN is able to retrotranspose.

As mentioned in the introduction, L1 elements usually act in cis, i.e. L1 proteins process predominantly the RNA molecule they are encoded on. This effect is probably due to spatial proximity of the RNA and the nascent protein, rather than recognition of specific sequences on the RNA. In rare cases, however, RNA from a retrotransposition-incompetent L1 element is retrotransposed by proteins derived from an active L1, a process known as trans complementation. Accordingly, fortuitous retrotransposition of foreign RNAs has been observed in an artificial cell culture system at a very low frequency (0.2-0.9% of wild-type L1 activity), but only if trans-acting L1 elements are highly over-expressed (Wei et al., 2001).

Since the low level of colony formation caused by the L1/Tx1L EN chimera pNZ39 ranged in the same order of magnitude, it was necessary to perform controls to address the question whether the observed transposition frequency is merely the consequence of trans complementation of an inactive L1 chimera.

In order to control for a possible trans complementation of RT activity by endogenous L1 elements, the control plasmid pNZ49, a pNZ39 analogue carrying the RT missense mutation D702A, was generated. This construct exhibited no retrotransposition potential (0.02%

relative to wild-type L1), showing that trans complementation of the RT does not occur.

Nevertheless, a further control experiment was performed to test whether the EN is indeed responsible for the observed retrotransposition events. For this purpose, construct pNZ51 was generated, carrying the same chimeric L1/Tx1L element as pNZ39 except that His 230, a residue involved in catalysis, is replaced by Ala. This point mutation had been used before to destroy L1 EN activity (Wei et al., 2001). The resulting mutated chimera was expected to be unable to transpose due to its inactivated EN. However, pNZ51 retained the same retrotransposition frequency as the parental construct pNZ39 (0.27 ± 0.07%, Fig. 17).

This particular histidine residue (H309 in APE1) is not directly involved in the catalytic cleavage of the scissile phosphate bond, but is one of three amino acids that fix the phosphate in the correct orientation (Mol et al., 2000). As it is highly probable that the conformation of the chimeric L1/Tx1L EN differs at least slightly from L1 EN or Tx1L EN, it was reasoned that H230 might not be a critical residue in the chimera and therefore indifferent to

mutations in the conserved residues D143 (the actual catalytic residue activating the attacking nucleophile), N145 and D205. The combination of mutations in the resulting constructs pNZ63-66 are indicated in Fig. 17.

Each point mutation had the same effect and did not result in a reduced retrotransposition frequency. Fig. 17 summarises the results obtained and illustrates that the changes of activity of the different mutants relative to pNZ39 are not statistically significant. Hence it must be concluded that the chimeric L1/Tx1L EN of pNZ39 itself is already inactive.

This result was rather unexpected. In an attempt to explain how the L1/Tx1L chimeras can transpose without a functional EN, the following theory was formulated: pNZ39 may be able to initiate retrotransposition without possessing a nucleolytically active EN because the chimeric EN can still bind to pre-existing nicks in the target DNA. It could thus recruit the element's ribonucleoprotein particle to the target DNA and initiate transposition with a higher frequency than for example the corresponding L1/R1 chimera pNZ33 (Fig. 16, p.66). If this were the case, an increased number of chromosomal nicks in the target cell should result in an augmented retrotransposition rate.

To test this hypothesis, the retrotransposition assay was performed under conditions that increased the amount of single-strand breaks in the host cell. Hydrogen peroxide (H2O2) is an oxidative reagent known to induce mainly single-strand DNA breaks when added to the medium of cultured cells (Dahm-Daphi et al., 2000). First, HeLa cells were titrated with H2O2

in order to establish the maximum concentration not harming the cells under the assay conditions (10^-5 M, causing an estimated 10⁴ single-strand breaks/cell, data not shown).

Subsequently, the L1/Tx1L EN chimera pNZ39 and its RT-mutant pNZ49 were tested in the retrotransposition assay in the presence or absence of 10^-5 M H2O2. The results of this experiment are shown in Fig. 18. While the retrotransposition frequency of pNZ39 was indeed elevated by a factor of 2.5 under the influence of H2O2, the same was true for pNZ49. This latter construct, however, should not react to an increase of genomic nicks as its RT is inactivated. Increased retrotranspositional activity in response to H2O2 treatment was also observed for pJM101/L1.3. This effect can be ascribed to transcriptional upregulation of L1 elements as a result of irradiation (Servomaa and Rytömaa, 1990) or oxidative stress (G. Tolstonog, Heinrich-Pette-Institut, personal communication), leading to subsequent increased trans complementation. Due to this general effect, it was not possible to identify a pNZ39-specific effect of single-strand breaks.

RESULTS 71

pNZ39

pNZ49

pJM101 H₂O₂

− +

− +

Retrotransposition frequency relative to pJM101 in the absence of H₂O₂ [%]

0.36±0.07 0.84±0.09 2.3

0.06±0.06 0.15±0.03 2.5

100±14.8 200±50 2.0 x-fold increase H₂O₂ H₂O₂

Transfected construct

Fig. 18: Treatment of HeLa cells with the oxidative reagent H₂O₂ leads to an increased retrotransposition frequency in both mutant and wild-type L1 elements. Representative results of retrotransposition assays performed with the indicated reporter constructs in the absence or presence of H₂O₂. Schematic drawings of pNZ39, pNZ49 and pJM101 are shown in Fig. 17. Relative retrotransposition frequencies were normalised for pJM101/L1.3 activity in the absence of H₂O₂ (n=3).

“x-fold increase” quantifies the stimulating effect of H₂O₂ treatment on retrotransposition.

3.3.2.3 An L1 element bearing the R1α8-helix is retrotransposition competent, while