PacI
Created by: Jack Allan
PacI is a homing restriction endonuclease belonging to the HNH structural family, named for the pair of histidine residues and single asparagine residue making up the zinc domains. In general, restriction endonucleases are a group of proteins believed to function in host genome defense against foreign DNA insertion, namely against bacteriophage invasion (1). More specifically to PacI, homing endonucleases are highly specific and in much the same way as transposons, insert their own open reading frame into recognition target sequences, disrupting transcription of foreign DNA while also perpetuating their own expression (2). PacI recognizes a target sequence 5′-TTAATTAA-3′ and is currently the smallest identified restriction endonuclease (1). PacI is considered a “rare cutting” endonuclease, much like NotI and SfiI, which also recognize infrequent DNA sequences but belong to the PD…(D/E)×K catalytic site superfamily. The structure of PacI (PDB ID: 3M7K) was elucidated in Pseudomonas alcaligenes.
The structure of PacI includes a polypeptide subunit of 142 amino acids with two associated DNA chain polymers of ten and eight base pairs. The secondary structure of PacI consists of five alpha helices made up of sixty residues and comprising forty-two percent of the overall protein structure as well as four beta strands made up of twenty residues and comprising twelve percent of the total protein structure.
The catalytic feature of the HNH motif endonucleases is termed the ‘‘ββα-metal’’ fold, a secondary structure consisting of two antiparallel β-strands connected by a loop sequence and flanked by an α-helix (1). For PacI specifically, the ββα-metal fold exists at β3-β4-α4 of the core topology, β1–β2–α2–α3–β4–α4–α5 (1). The ββα-metal fold is generally complexed with a magnesium ion and although calcium can also exist in this complex, there exists a free 5’ phosphate and 3’ hydroxyl DNA group exposed when magnesium is present in complex. The PacI subunit also contains two bound zinc ions, one surrounding the ββα-metal fold and both of which are coordinated with four cysteine residues each. One of the zinc ions is embedded within the N-terminal of the polypeptide chain while the second is embedded within the core of the protein and is coordinated with the ββα-metal fold via two cysteine residues contained within the 4α-helix (1).
The PacI ββα-metal domain contains several major alterations in primary sequence from the canonical HNH domain. Most significantly, the functional His residue of other HNH endonucleases, the general base that functions to activate water as a nucleophile in the displacement of the 3’ DNA leaving group during endonuclease activity, is replaced in PacI by Arg-93. In Hpy99I, the similar HNH homing endonuclease previously mentioned, the His-149 residue is crucial in the activation of water toward activity of the catalytic site, thus the presence of the Arg-93 alteration for this same function is evidence of PacI’s divergence from other HNH homing family endonucleases (3). An Arg-93 to Asn-93 leads to compromised PacI function, evidencing the importance of this residue in PacI despite its structural difference from other HNH family endonucleases (1),(3).
Both Tyr-100 and His-42 of PacI maintain seemingly crucial functions not relevant in other HNH ββα-metal motifs. The Tyr-100 residue apparently maintains a function toward assisting in water activation or in acting as a nucleophile itself. Point mutations of Tyr-100 to Phe-100 and His-42 to Lys-42 produced a PacI protein with compromised function. The metal-binding residues, Asp-92 and Asn-113, also showed functional importance in PacI activity and point mutations of these residues produced decreased functionality although the importance of these residues is not fully understood (1). The extent of the structural divergence of PacI in its ββα-metal motif could potentially be indication of an alternate cleavage mechanism at work. In fact, other similar divergence from the canonical HNH structure has led some researchers to suggest that the title of "ββα-motif structural family" would more generally reflect the cleavage mechanism at work in these endonucelases (4).
In its functional form, PacI is an extended homodimer and achieves its endonuclease activity when the ββα-metal catalytic sites of each subunit associate with and straddle the minor groove of the target DNA sequence. This interaction leads to a widening of the minor groove and narrowing of the major groove, ultimately causing the dissociation of the hydrogen bonds between each Watson-Crick pair in the target sequence. The base pairings between each DNA strand are thus subsequently rearranged, leaving two base pairs unpaired, four base pairs in non-canonical pairings, and two base pairs with new Watson-Crick pairings. DNA strand cleavage is achieved as the rearrangement of the DNA molecule leads to a distortion of the glycosidic bonds in the target region (1).
The protein-DNA interactions of PacI with it’s target sequence involves only eight hydrogen bonds in each DNA half site: the unpaired A and T interact in the minor groove with Asn-32 and Arg-114, respectively; the A bases reorganized in the A:T pairings bond with Asn-36 in the major groove; one A in each non-canonical A:A bond interacts with Ser-117, also in the minor groove; and the O4 groups of T in each T:T are hydrogen bonded to Lys-39 in the major groove. The importance of these amino acid-base pair interactions is confirmed via mutation of Asn-32 and Asn-36, which lead to loss of PacI function. However, mutations to Lys-39 seemed to have no effect on function, leading to the assumption that this minor groove bond is less important. Because hydrogen bonds are thought to be the most important protein-DNA bond type relevant to recognition specificity, the few number of hydrogen bonds found in the PacI-DNA complex has provided some challenge in understanding the protein’s target sequence specificity (5),(1).
PacI is largely unique in its structure, with no evidence of conserved regions through BLAST analysis. However, review of current literature reveals that PacI superficially resembles the homodimeric HNH restriction endonuclease Hpy99I, and even more superficially the homodimeric HNH homing endonuclease I-PpoI. The common features of the three proteins include a catalytic ββα-metal motif, two structural zinc ions embedded within the protein subunit, and active sites across the minor groove to produce 3′ overhangs. The ultimate extended structure of these proteins, however, are all very distinct, indicating a divergence from a common ancestral protein followed by derivation of a highly specialized structure specific to each protein’s function (1).
Hpy99I and I-PpoI both have similar genome protection functions as PacI, although acting on different recognition sequences. PacI is particularly interesting, and worthy of study, as it is not paired with a companion methyltransferase protein, as is the case with most other endonucleases. This is of interest because the methyltransferase protein would generally function to protect the host genome from PacI’s activity, thereby protecting the host genome from PacI-induced degradation. This lack of companion methyltransferase implies that PacI’s recognition sequence is absent from the host genome, indicating that PacI’s structure and functions are highly specialized (1).