It is likely that the grouping of at least the Tetrahymena proteins into this clade is a result of convergent evolution of mART activity. Given kinase inhibitor Afatinib the heterogeneous composition of Clade 3, it is difficult to divide into subclades, however, we classified the proteins into six subclades as outlined below, par tially for the purpose of discussion, and partially based on common domain structures and features of the cata lytic domains. Clade 3A is composed of two proteins, including human PARP10, containing an RRM RNA binding domain, a glycine rich region, and a UIM domain, known to bind monoubiquitin and polyubiquitin chains. The proteins found in Clade 3B and 3C contain at least one Macro domain N terminal to their C terminal cata lytic domain. Macro domains have been shown to bind to poly.

Clade 3B includes representatives from the most basal animal in our study Trichoplax adhaerens, while 3C includes two human proteins, PARP14 and PARP15. PARP10, PARP14 and PARP15 have been demonstrated to have mART activity. Clade 3D consists of the two Dictyostelium discoideum and four Tetrahymena thermophila proteins. Unlike the majority of animal proteins in Clade 3, only one of these proteins have a proline located one amino acid away from the third residue of the catalytic triad. The four proteins from the ciliate Tetrahymena thermo philia have no known functional domains outside of their C terminal PARP catalytic domains and are only similar to one another in this region, again supporting the idea that these proteins are not closely evolutionarily related to the other proteins in Clade 3.

One of the Tetrahymena proteins has retained the glutamic acid of the HYE, again sup porting this interpretation. All four proteins also share a H NNSK motif just past the last amino acid of the puta tive catalytic triad not found in other members of Clade 3. The Dictyostelium proteins in 3D do not show high similarity outside of the PARP domain. DDB0304590 is a relatively short protein with only the PARP catalytic domain and a short C terminal exten sion. DDB0232928 has a Macro domain and, at its very N terminus, a U box. The U box is a modified RING finger found in E3 ubiquitin ligases known to bind ubiquitin E2 enzymes. As Amoebo zoa is the sister group to Opisthokonts within eukar yotes and given that DDB0232928 contains a Macro domain as do some other members of Clade 3, it is pos sible that these proteins are orthologous to at least some of the animal Clade 3 proteins.

Clade 3E is confined to animals, but is not represented in Placozoa. Members of this subclade con tain one to two WWE domains, alone or in combination with zinc fingers in front of their PARP catalytic domains. All members of 3E have replaced the glutamic acid characteristic of PARPs with an isoleucine except for two that con tain valines at GSK-3 that site. This subclade also contains human PARP12 and human PARPT PARP7.