The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain.

Nickens Nickens,Sausen Sausen,Bochman Bochman

doi:10.3390/genes10060411

Abstract

Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

Highlights

DNA helicases are enzymes that couple DNA binding and ATP hydrolysis to unwind double-stranded DNA into its component single strands [1]
Cell growth did not completely recover to levels observed in the absence of Pif1 overexpression (Pif1 glucose vs. Pif1∆N galactose, p = 0.0002), we still found that the toxicity of overexpression was significantly relieved in the absence of the Pif1 N-terminal domain (NTD) (Pif1 galactose vs. Pif1∆N galactose, p < 1.2 × 10−6 ; Figure 1A)
To begin to determine the roles of the non-helicase domains of Pif1, we compared the activity of full-length Pif1 and a Pif1 truncation lacking the entirety of the natively disordered NTD (Pif1∆N)

Summary

Introduction

DNA helicases are enzymes that couple DNA binding and ATP hydrolysis to unwind double-stranded DNA (dsDNA) into its component single strands [1] This activity is vital to many processes involved in the maintenance of genome integrity, including DNA replication, recombination and repair, transcription, and telomere maintenance [2]. Some helicases in the RecQ family contain an exonuclease domain (e.g., the Werner syndrome helicase WRN [5]), a DNA strand exchange domain (e.g., RecQ-like helicase 4, RECQL4 [6]), and/or domains involved in DNA and protein interactions (RecQ C-terminal domain (RQC) and helicase and RNaseD C-terminal domain (HRDC), respectively) [7] These accessory domains may be used in tandem with helicase activity to ensure genome integrity, or they may be functionally separable. The latter is the case with RECQL4, which has an N-terminal domain (NTD) necessary for the initiation of DNA replication with no dependence on helicase activity [8]

Methods

Results

Conclusion