DNABinding Domain Diversity and Conserved Modules

There appears to be very little sequence similarity outside of the GIY-YIG module among the members of the GIY-YIG family, except among proteins closely related to I-TevI, like I-Bmol, an intron-encoded enzyme from Bacillus mojavensis (Edgell and Shub 2001). I-TevI and I-Bmol can be aligned through their GIY-YIG domains and through parts of their DNA-binding domains. I-Bmol has a H-T-H domain analogous to that of I-TevI, but it lacks a Zn-fin-ger domain. Instead, it appears to have three copies of the minor groove-binding a-helix present in I-TevI (Sitbon and Pietrokovski 2003).

However, a number of short stretches of conserved sequence have been identified, through multiple sequence alignments (Sitbon and Pietrokovski 2003), that are found in many GIY-YIG proteins and are also shared with certain HNH endonucleases and other proteins. In most cases, the structural and functional significance of these sequence segments is unclear, but one such conserved domain (NUMOD 3; Sitbon and Pietrokovski 2003) corresponds to the minor groove-binding a-helix present in the DNA-binding domain of I-TevI, suggesting that this might be conserved among many of the otherwise divergent proteins. Indeed, NUMOD 3 is predicted to be present in several GIY-YIG enzymes, including four of the Seg proteins. Interestingly, it is present in multiple copies in several other proteins, besides I-Bmol, suggesting the presence of multiple DNA-binding a-helices.

The modular nature of I-TevI has led us to propose that the GIY-YIG catalytic cartridge can acquire specificity by associating with various DNA-bind-ing domains (Derbyshire et al. 1997). The structure of the DNA-binding domain of I-TevI, and comparisons with the sequences of other family members, show this to be the case. This modular organization that separates cleavage and DNA binding into distinct domains is a feature shared with the HNH proteins but not with the LAGLIDADG and His-Cys box endonucleases, which have their catalytic and DNA-binding functions within a single subunit or protein domain. The structure of I-Hmul, an HNH homing endonuclease encoded by a group I intron in the B. subtilis bacteriophage SPOl, has recently been solved (Shen et al. 2004). What is most remarkable about this structure is that, despite the fact that I-Hmul and I-TevI are from unrelated endonuclease families, their DNA-binding domains have very similar structures (Fig. 6). I-Hmul has a C-terminal H-T-H domain and two copies of a minor groove-binding a-helix. The structures of the H-T-H domain and of the second a-helix resemble those of I-TevI very closely. However, the protein-DNA interactions carried out by these subdomains are actually somewhat different in the two proteins. Consistent with its high sequence tolerance, I-TevI interacts primarily with the phosphate backbone and ribose moieties whereas I-Hmul, which is much more sequence specific, makes many contacts directly with the bases. The a-helix of I-Hmul is bound much more deeply into the minor groove of its target than that of I-TevI. This causes a significant distortion of the DNA, whereas the I-TevI homing site is largely unperturbed.

It would thus appear that the DNA-binding modules are shuffled with different endonuclease cartridges, and that each then adapts to the particular biology of the organism in which the enzyme resides. On one hand, the mosaicism of these endonucleases and their genes provides evidence for modular exchange and therefore rapid evolution. On the other hand, adaptations to

Fig. 6. Three-dimensional structure of I-Hmul with its substrate (Shen et al. 2004). The H-T-H domain and the second minor groove-binding a-helix are very similar, both in structure and relative orientation to the corresponding segments of I-TevI (see Fig. 3A)

Fig. 6. Three-dimensional structure of I-Hmul with its substrate (Shen et al. 2004). The H-T-H domain and the second minor groove-binding a-helix are very similar, both in structure and relative orientation to the corresponding segments of I-TevI (see Fig. 3A)

their specific host ensure the enzyme's persistence. This combination of promiscuity and restraint is a hallmark of successful parasitic elements.

Acknowledgements. We thank Maryellen Carl for expert secretarial assistance, John Danse-reau for preparing the figures, and Joe Kowalski for preparing the LOGOS figure. This work is supported by NIH grant GM44844 to Marlene Belfort.

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