The S. cerevisiae PIF1
gene was isolated in a genetic screen for genes that affect the frequency of mitochondrial DNA (mtDNA) recombination between wild type and cytoplasmic petite mutant strains (i.e.
]. A decade later, the same group succeeded in partially purifying ScPif1 from mitochondria and demonstrated that it possesses single-stranded DNA (ssDNA)-dependent ATPase and 5′→3′ directed helicase activities [2
]. Soon after, the study of Pif1 helicases as proteins affecting the nuclear genome began with the “rediscovery” of ScPif1 as an enzyme that inhibits telomere elongation and de novo
telomere formation [3
] (see Section 3
for an in-depth discussion of ScPif1).
A second PIF1
-like gene, now called RRM3
, was found in S. cerevisiae
by two different groups. In one case, the gene was identified by searching the database for genes with similarity to ScPIF1
]. In the second, it was identified in a screen for r
ibosomal DNA (rDNA) r
], where it was named ScRRM3
]. As more genome data became available, it became apparent that ScPif1 was the founding member of a family of helicases conserved in essentially all eukaryotes (). However, while several fungal genomes, such as Candida albicans
and Cryptococcus neoformans
, are like S. cerevisiae
in encoding two Pif1 family helicases, most higher eukaryotes and all metazoans contain only one. Two notable exceptions to this rule are Arabidopsis thaliana
with three Pifs () and kinetoplastid parasites with seven to eight Pif1 helicases (see Section 7
). Regardless of the number of Pif1-like proteins in an organism's genome, all of the Pif1 family proteins are comprised of a conserved 400-500 amino acid ATPase/helicase domain but have divergent N- and C-termini ().
Figure 1 Evolutionary relationship among Pif1 family and RecD helicases. The indicated sequences were aligned using ClustalX , and the phylogenetic relationship among them was drawn as a rooted tree (using the unrelated human beta actin protein (NP_001092) (more ...)
Figure 2 Conserved motifs in the Pif1 family helicases. The sequences of the Hs-, Mm-, and ScPif1, ScRrm3, SpPfh1, and TbPif5 helicases used to generate were aligned using ClustalW , and the BOXSHADE program in the Biology WorkBench suite (http://workbench.sdsc.edu (more ...)
Originally, proteins with high similarity to Pif1 helicases were found only in eukaryotes, although BLAST searching [6
] for homologues of ScPif1 revealed that it is distantly related (16.4% identity) to the Escherichia coli
RecD helicase [7
]. A current search of the NCBI protein database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=Protein&itool=toolbar
) reveals that several putative prokaryotic proteins are annotated as Rrm3-/Pif1-like (see and legend). To determine if these sequences are evolutionarily related or simply related by being ATPases/helicases, we constructed a phylogenetic tree of prokaryotic RecD (and Rrm3-/Pif1-like) proteins and eukaryotic Pif1 and Rrm3 proteins. As shown in , the prokaryotic Bdellovibrio bacteriovorus, Campylobacter jejuni,
and Clostridium sporogenes
Rrm3/Pif1 proteins do cluster with eukaryotic Pif1 family members as opposed to RecD proteins. It should be noted, however, that these Rrm3-/Pif1-like sequences are grouped with some of the most divergent eukaryotic Pif1 family helicases (e.g., C. sporogenes
Rrm3/Pif1 forms a clade with the Pif8 homologues from parasites, which are believed to have lost their helicase activities (see Section 7.4
). Also, the Agrobacterium radiobacter
RecD appears to be closely related to the Pif1 helicases from plants. In all, these results suggest that RecD, Rrm3, and Pif1 may have evolved from a common proto-helicase.
Regardless of ancestry, the evolution of Pif1 helicases is intriguing. At some point in the past, there existed a single progenitor helicase that evolved into two helicases with distinct functions in fungi (and seven or more in parasites), the most-widely characterized duo being ScPif1 and ScRrm3 (it should be noted that the presence of two Pif1 helicases does not appear to be due to the ancient genome duplication that occurred in S. cerevisiae (J. Bessler, unpublished)). Then, in most eukaryotes, one of these enzymes was lost, resulting in the single Pif1 family helicase found in metazoans. The current situation leaves us with several questions, including whether ScRrm3 or ScPif1 is more closely related to the ancestral form and if metazoan Pif1 helicases are more similar to ScPif1 or ScRrm3. Based on the phylogeny presented in , it is difficult to address either of these questions definitively, but the branch lengths of the fungal Pif1 and Rrm3 clades indicate that ScRrm3 may have evolved first.
Questions of evolution aside, the Pif1 family belongs to the super-family IB helicases, which are comprised of mostly monomeric, 5′→3′ directed, P-loop (a conserved nucleotide binding motif, also known as “the Walker A box” or “motif I”) helicases [9
]. This view comes from an analysis of Pif1 sequence alignments () that shows the seven conserved SFI motifs (I, Ia, II, III, IV, V, and VI) and the three motifs shared with E. coli
RecD (A, B, and C). There is also a putative 21-residue Pif1 family signature sequence located between motifs II and III. This motif is highly conserved in 24 of the 33 (72.7%) eukaryotic Pif1 family proteins used to generate the phylogenetic tree in (M. Bochman, observations) but is degenerate or absent in the plant Pif1 helicases and the parasitic Pif1, 2, 4, and 8 homologues, which are the most divergent sequences in the Pif1 family alignment. This motif is also absent in RecD homologues. Finally, using the consensus sequence for this motif (DKLeXvARaiRkqXkPFGGIQ) to query the NCBI protein database via
BLAST searching [6
] returns only Pif1 homologues (M. Bochman, observations).