Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

Abstract

Pif1 helicase plays various roles in the maintenance of nuclear and mitochondrial genome integrity in most eukaryotes. Here, we used a proteomics approach called isotopic differentiation of interactions as random or targeted to identify specific protein complexes of Saccharomyces cerevisiae Pif1. We identified a stable association between Pif1 and a mitochondrial SSB, Rim1. In vitro co-precipitation experiments using recombinant proteins indicated a direct interaction between Pif1 and Rim1. Fluorescently labeled Rim1 was titrated with Pif1 resulting in an increase in anisotropy and a Kd value of 0.69 µM. Deletion mutagenesis revealed that the OB-fold domain and the C-terminal tail of Rim1 are both involved in interaction with Pif1. However, a Rim1 C-terminal truncation (Rim1ΔC18) exhibited a nearly 4-fold higher Kd value. Rim1 stimulated Pif1 DNA helicase activity by 4- to 5-fold, whereas Rim1ΔC18 stimulated Pif1 by 2-fold. Hence, two regions of Rim1, the OB-fold domain and the C-terminal domain, interact with Pif1. One of these interactions occurs through the N-terminal domain of Pif1 because a deletion mutant of Pif1 (Pif1ΔN) retained interaction with Rim1 but did not exhibit stimulation of helicase activity. In light of our in vivo and in vitro data, and previous work, it is likely that the Rim1–Pif1 interaction plays a role in coordination of their functions in mtDNA metabolism.

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

“…Therefore, the average distance traveled by Pif1 equal to 1/(1-P) is 10 bp of duplex on average before dissociation. This value is associated with relatively nonprocessive helicases, and it is consistent with previous results (19,21,23).…”

“…Recombinant Proteins-Pif1 was purified as described (23). Cells expressing the E. coli A197C phosphate-binding protein (PBP) were grown as described (25), and the A197C-PBP was purified and labeled with MDCC as described (26).…”

“…In vitro telomerase assays showed that the presence of Pif1 reduces the processivity of the telomerase complex (9). A recent report showed that Pif1 stably interacts with a mitochondrial SSB, Rim1, both in vivo and in vitro (23). The presence of Rim1 in helicase assays enhanced Pif1 unwinding activity.…”

Linkage of Oxidative Stress and Mitochondrial Dysfunctions to Spontaneous Culture Degeneration in Aspergillus nidulans

“…The putative DNA helicase AN5278 and DNA repair nuclease AN3118 were only detected in sector (supplemental Table S2) and are the homologs of yeast Pif1 (26%) and Mus81 (36% identity), respectively. Pif1 helicase has been shown to play important roles in the maintenance of both nuclear and mt genome integrity by interacting with a single-strand binding protein (63). Mus81 is shown to serve as a subunit of a heterodimeric nucleases complex that efficiently repairs DNA by resolving DNA intermediates (64).…”

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

“…Mitochondrial SSB is known to stimulate DNA helicase activity (96). Indeed, Rim1 stimulates Pif1 activity by 4-to 5-fold, and these two proteins physically interact with each other (103). In the absence of a functional Pif1 helicase, the suppression of the respirationdeficient phenotype by Rim1 also suggests that the latter may stimulate the activity of a second DNA helicase that functionally overlaps with Pif1.…”