The catalytic activities of Tf1 IN and the inhibitory function of its chromodomain (Hizi and Levin 2005).

The IN of Tf1 contains a Zn finger-like motif and a DDE motif, two sequences typical of retroelement integrases (Levin, Weaver et al. 1990).  Further analysis of its sequence revealed that Tf1 IN also contains a chromodomain (CHD) at its C-terminus (Malik and Eickbush 1999).  CHDs are present in chromatin modifying proteins such as Hp1 and interact directly with histone proteins with specific post-translational modifications.  The presence of a CHD suggests Tf1 IN may interact directly with specific nucleosomes during integration.  This is particularly interesting in the case of Tf1 because such an interaction may play a role in the insertion preference of Pol II promoters.  In these first studies of the CHD, we examined its role in the catalytic activities of IN. 

1.  The catalytic activity of IN is inhibited by the CHD.

The full-length IN and IN lacking the CHD (CH-) were expressed in bacteria and purified extensively with cobalt agarose and heparin sepharose.  To measure catalytic activity and optimize reaction conditions we used the disintegration assay.  This assay measures the reverse of integration and was used initially because the substrate sequence and structural requirements are less stringent than for the forward reaction. The model substrate consisted of a 76 nt oligonucleotide that mimics the end of the LTR attached to the bottom strand of a target. A 20 nt oligonucleotide is added to mimic the top strand of the target. IN activity will catalyze a transesterification reaction that joints the 20 nt top substrate to the bottom strand of the target.  This converts the 20 nt DNA into a 63 nt product, a change that can be readily detected on polyacrylamide gels. These experiments revealed that IN possessed substantial levels of catalytic activity.  Surprisingly, the CH- protein was significantly more active then the full length IN.

To test the IN proteins for their ability to catalyze the forward reaction, a 24 nt double-stranded DNA was used as the substrate in strand transfer assays.  The DNA contained the sequence of either the U3 or U5 ends of the Tf1 LTR and served both as donor and target.  IN exhibited substantial strand transfer activity as indicated by the production of higher molecular weight DNAs.  Once again, the IN lacking the CHD, CH-, had substantially more activity than the full-length IN.  These data substantiated that Tf1 IN was active as a recombinant protein and that the CHD functioned to inhibit this activity.

The strand transfer activities of retrovirus and retrotransposon INs require that the highly conserved “CA” dinucleotide be present at the 3’ end of the donor substrate.  We tested whether the IN of Tf1 had this same requirement for “CA” and whether the CHD contributed to this specificity. IN and CH- were tested for whether each of the last three nucleotides of the LTRs, “ACA” was necessary for strand transfer. All the modifications introduced in the sequence led to a dramatic reduction of the strand transfer activity of IN. In sharp contrast to IN, CH- retained high levels of activity with most of the substituted substrates. For example CH- retained 50% of its activity with the substrate that had a minus one transition (AtA) whereas the full length IN had just 1% of its activity with this substrate. This large difference in the specificity of the two related proteins shows that IN was far more stringent than the CH- counterpart in selecting the DNA donor with the correct 3’ end.

Both contributions of the CHD, limiting IN activity and increasing sequence specificity, may result from a single mechanism that restricts access to the active site of IN. One intriguing hypothesis is that the CHD restricts IN activity until it interacts with histone H3 or some other factor and it is this interaction that relieves the inhibitory function of the CHD and allows integration to occur.

2.  Tf1 IN possesses 3’ processing activity.

The INs of retroviruses have a 3’ processing activity that removes the two or three nucleotides 3’ of the “CA” so that strand transfer can occur. The primer for the minus strand of Tf1 is positioned adjacent to the LTR allowing the cDNA to terminate with the “CA” that is necessary for strand transfer. Thus it was predicted that no processing activity would exist (Levin 1995; Levin 1996).  However, sequences of Tf1 cDNA extracted from particles revealed the surprising result that 85% of the 3’ ends had one or more untemplated nucleotides (Atwood-Moore, Ejebe et al. 2005). Because these nucleotides are expected to block strand transfer, we tested whether Tf1 IN had a processing activity. In this experiment, the 5’-end labeled substrates contained nucleotides positioned 3’ to the critical “CA”.  The data from these experiments showed that IN did have processing activity capable of removing as many as 5 nucleotides from the 3’ end of the substrate. The processing activity could in theory remove the 3’ nontemplated nucleotides and allow the bulk of the cDNA to participate in integration.  This model raises the question why would nontemplated nucleotides be added just so they could be removed by IN.  One possibility is that the nontemplated nucleotides could protect the conserved “CA” from attack by nonspecific 3’ exonucleases.