Identification of Pif1 helicases with novel accessory domains in various amoebae

https://doi.org/10.1016/j.ympev.2016.07.015Get rights and content

Highlights

  • We have identified ten amoebal Pif1 helicases possessing novel accessory domains.

  • Domains are involved in ubiquitination, DNA replication or nucleic acid binding.

  • These proteins evolved via horizontal gene transfer, gene duplication and fusion.

  • Acanthamoeba accessory domains were acquired from fungi in two different phyla.

  • These domains have likely improved or expanded the roles of amoebal Pif1 helicases.

Abstract

Pif1 helicases are a conserved family of eukaryotic proteins involved in the maintenance of both nuclear and mitochondrial DNA. These enzymes possess a number of known and putative functions, which facilitate overall genome integrity. Here we have identified multiple subtypes of Pif1 proteins in various pathogenic and non-pathogenic amoeboid species which possess additional domains not present in other Pif1 helicases. These helicases each possess one of five different accessory domains, which have roles in ubiquitination, origin of DNA replication recognition or single-stranded nucleic acid binding activity. Using a robust phylogenetic approach we examined each Pif1 class, which revealed that gene duplication, fusion and horizontal gene transfer events have all contributed to the evolution of these enzymes. This study has identified the first collection of Pif1 helicases to contain additional domains, which likely confer novel enzymatic activity, or improve existing functionality. Furthermore, the potential functions of these helicases may shed further light on the overall role the Pif1 family plays in genome maintenance.

Introduction

The proper maintenance of DNA is of critical importance for normal cellular function. While a variety of proteins are involved in DNA maintenance, helicases are required for most of these processes due to their ability to unwind duplex nucleic acid structures. One of the major eukaryotic DNA helicase families involved in genome maintenance is the Pif1 superfamily, which was originally identified in Saccharomyces cerevisiae while screening for genes affecting mitochondrial DNA recombination (Foury and Kolodynski, 1983). These helicases are of particular interest due to their role in maintaining both nuclear and mitochondrial DNA within cells through a variety of pathways. The budding yeast and human Pif1 helicases (ScPif1 and hPif1, respectively) localize to both the nucleus and mitochondria (Futami et al., 2007, Lahaye et al., 1991, Zhou et al., 2000), although little is known about their roles in mitochondria beyond general maintenance and recombination (Cheng et al., 2007, Foury and Kolodynski, 1983). Conversely, the characterization of nuclear Pif1 has been more extensive.

Pif1 is a multifaceted helicase as its nuclear isoform has known and putative roles in a range of maintenance processes. hPif1 negatively regulates telomere length, as its overexpression results in telomere shortening (Zhang et al., 2006). This function is likely due to Pif1 being capable of inhibiting telomerase activity or removing it from telomeres (Boulé et al., 2005, Zhang et al., 2006, Zhou et al., 2000). This enzymatic activity is also important for proper DNA repair, as the inhibition of telomerase prevents telomeres being added to double strand breaks (Schulz and Zakian, 1994), which would otherwise lead to chromosomal deletion. It has been suggested that this inhibition is due to the removal of telomeric RNA, given that Pif1 has a preference for unwinding RNA-DNA substrates (Boule and Zakian, 2007).

Pif1 is also able to resolve G-quadruplex (G4) structures which would otherwise impede DNA replication. G4s form when four DNA sequences rich in guanine anneal together, which may be inter- or intramolecular (Bochman et al., 2012). While replicative helicases cannot proceed through these structures (Bharti et al., 2014), Pif1 is able to efficiently unwind them and allow the continuation of DNA replication (Paeschke et al., 2011, Sanders, 2010). Furthermore, Pif1 is able to load itself onto 3′ ends of single-stranded DNA, during which it can unwind RNA-DNA hybrids and intramolecular G4s to potentially allow for normal progression of DNA replication, repair and recombination (Zhou et al., 2014).

Pif1 is theorized to aid the joining of Okazaki fragments during DNA replication of the lagging strand. DNA polymerase δ synthesizes the lagging DNA strand from an RNA primer and extends until it meets a downstream primer, which typically produces a short flap, allowing for primer removal. However, Pif1 is thought to promote the lengthening of this flap, which then requires an alternate removal process, likely to include the Dna2 helicase/nuclease (Rossi et al., 2008). This is supported by the fact that pif1 deletion in yeast rescues the otherwise lethal deletion of dna2 (Budd et al., 2006). This rescue is likely a result of Pif1 no longer promoting Dna2 requirement, driven by its ability to displace DNA polymerase δ (Pike et al., 2009).

Pif1 helicases may also perform other functions in DNA replication beyond lagging strand synthesis. S. cerevisiae ribosomal DNA is flanked by replication fork barriers, which limit replication progression to a single direction to prevent the replication and transcription machinery from colliding. S. cerevisiae possesses two Pif1 homologues, ScPif1 and ScRrm3, which play opposing roles in maintaining these barriers, as their knockout results in either improper arrest or progression of replication at the barrier, respectively (Ivessa et al., 2000). Furthermore, ScRrm3 may play a global role in replication of the nuclear genome, as it has been shown to associate with replication forks, a polymerase subunit of the replisome (Azvolinsky et al., 2006) and proteins involved in recognition of DNA replication origins (Matsuda et al., 2007). hPif1 is also involved in reparative DNA synthesis, as it stimulates both homologous recombination and break-induced repair by DNA polymerase δ following DNA breakage (Wilson et al., 2013).

Although Pif1 is present in most eukaryotes, the number of homologues can vary between different lineages. Metazoans, such as Homo sapiens, appear to only encode a single Pif1 helicase, while fungi and yeasts may also encode the Rrm3 homologue (Bochman et al., 2010, Zhou et al., 2000). The most disparate organisms studied are the kinetoplastids, such as Trypanosoma brucei, which can contain up to eight Pif1 proteins that appear to each be targeted to a single subcellular location (Liu et al., 2009). While no studies have investigated the Pif1 family in plants, in silico analysis of helicases in Arabidopsis thaliana suggests between three and eleven homologues could exist (Bochman et al., 2010, Knoll and Puchta, 2011).

The evolution of the Pif1 family has not been examined in great detail, although the number of homologues in several lineages suggests that gene duplication may have contributed to its expansion. Given the one or two Pif1 helicases in metazoans and fungi, respectively, it is hypothesized that the ancestral Pif1 was duplicated and subsequently lost in lineages such as the metazoans (Bochman et al., 2010). However, it is clear that further expansion has occurred in other lineages, such as plants and kinetoplastids. While this appears largely due to gene duplication, the presence of potential Pif1 homologues in prokaryotes (Bochman et al., 2010) suggests that horizontal gene transfer (HGT) may have also contributed to their evolution. Given that bacterial Pif1 helicases are more similar to eukaryotic Pif1 than other bacterial helicases (Bochman et al., 2011), it is likely that the expansion in certain lineages could include their spread via eukaryote-to-prokaryote HGT. While further exploration is needed, it is clear that the Pif1 family has been shaped by various genetic and evolutionary events.

To date there has been no in-depth analysis of Pif1 in amoebae; one cursory phylogenetic study identified two homologues in Dictyostelium discoideum (Bochman et al., 2010), although further examination of this species or other amoebae has not been reported. Here we present the first report investigating the distribution and evolution of Pif1 helicases in both pathogenic and non-pathogenic amoebae. We have identified a number of putative Pif1 homologues from amoebae which are unique compared to previously reported Pif1 helicases, as they not only contain the conserved familial helicase motif but also possess additional domains. The five different domains present in these helicases are capable of varying functions; this includes ubiquitination, recognition of the origin of DNA replication and single-stranded nucleic acid binding. Lastly, phylogenetic analysis of these novel accessory Pif1 helicases revealed that HGT, gene duplication and gene fusion have contributed to their evolution.

Section snippets

Sequence analysis

All helicases in this study were identified using Pif1 from D. discoideum as a query in the NCBI protein database (BLASTP). The domain structure of each putative accessory Pif1 helicase was determined using InterProScan (Jones et al., 2014). The subcellular localization of proteins was predicted using the consensus from a combination of software programs; NucPred (Brameier et al., 2007), Mitoprot (Claros and Vincens, 1996), Predotar (Small et al., 2004), TargetP (Emanuelsson et al., 2007) and

Identification and evolution of putative amoebal Pif1 helicases with additional domains

In this study we examined several amoeboid species to identify the prevalence and distribution of Pif1 helicases in these lineages. However, when examining these helicases for their domain architecture it became apparent that a number possessed domains which have not been previously reported in the Pif1 family. As a result, we investigated the evolutionary relationship of these proteins to elucidate their origin or putative function.

From a consortium of amoeboid helicases, we identified ten

Acknowledgements

AH was the recipient of a La Trobe University Postgraduate Research Scholarship. We wish to thank Sanja Aracic for advice with revision of the manuscript.

References (58)

  • B. Liu et al.

    Trypanosomes have six mitochondrial DNA helicases with one controlling kinetoplast maxicircle replication

    Mol. Cell

    (2009)
  • J. Méndez et al.

    Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication

    Mol. Cell

    (2002)
  • K. Paeschke et al.

    DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase

    Cell

    (2011)
  • J.E. Pike et al.

    Pif1 helicase lengthens some Okazaki fragment flaps necessitating Dna2 nuclease/helicase action in the two-nuclease processing pathway

    J. Biol. Chem.

    (2009)
  • T.A. Richards et al.

    Evolution of filamentous plant pathogens: gene exchange across eukaryotic kingdoms

    Curr. Biol.

    (2006)
  • M.L. Rossi et al.

    Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal

    J. Biol. Chem.

    (2008)
  • V.P. Schulz et al.

    The Saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formation

    Cell

    (1994)
  • A. Azvolinsky et al.

    The S. cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes

    Genes Dev.

    (2006)
  • S.P. Bell et al.

    ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex

    Nature

    (1992)
  • B.L. Bertolaet et al.

    UBA domains of DNA damage-inducible proteins interact with ubiquitin

    Nat. Struct. Mol. Biol.

    (2001)
  • T. Biederer et al.

    Role of Cue1p in ubiquitination and degradation at the ER surface

    Science

    (1997)
  • M.L. Bochman et al.

    The Pif1 family in prokaryotes: what are our helicases doing in your bacteria?

    Mol. Biol. Cell

    (2011)
  • M.L. Bochman et al.

    DNA secondary structures: stability and function of G-quadruplex structures

    Nat. Rev. Genet.

    (2012)
  • J.-B. Boulé et al.

    The yeast Pif1p helicase removes telomerase from telomeric DNA

    Nature

    (2005)
  • J.-B. Boule et al.

    The yeast Pif1p DNA helicase preferentially unwinds RNA–DNA substrates

    Nucl. Acids Res.

    (2007)
  • M. Brameier et al.

    NucPred – predicting nuclear localization of proteins

    Bioinformatics

    (2007)
  • M.E. Budd et al.

    Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase δ

    Mol. Cell. Biol.

    (2006)
  • C. Cayrou et al.

    New insights into replication origin characteristics in metazoans

    Cell Cycle

    (2012)
  • S.M. Cerritelli et al.

    Ribonuclease H: the enzymes in eukaryotes

    FEBS J.

    (2009)
  • View full text