The Pif1p subfamily of helicases: region-specific DNA helicases?

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The Pif1p subfamily of helicases: regionspecific DNA helicases? Jessica B. Bessler, Jorge Z. Torres and Virginia A. Zakian DNA helicases are required for DNA replication, recombination and repair. Despite a common enzymatic function – the ability to unwind duplex DNA – most helicases share only limited amino acid sequence similarity. Helicases that have significant sequence similarity define a subfamily. It remains unclear, however, how this sequence similarity relates to helicase function. The Saccharomyces cerevisiae Pif1p helicase is the prototype member of a helicase subfamily that is conserved from yeasts to humans. As the two Pif1p subfamily members studied to date affect the same DNA sequences, the amino acid similarity that defines this subfamily might reflect common substrates.

Jessica B. Bessler† Jorge Z. Torres† Virginia A. Zakian* Dept of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. *e-mail: vzakian@ molbio.princeton.edu †These authors contributed equally to this work.

Helicases harness the energy of NTP hydrolysis to catalyse the unwinding of duplex nucleic acids. As a result, helicases play crucial roles in essentially every function that involves DNA and RNA, including DNA replication, repair and recombination, and RNA transcription, processing and translation. Their wide range of functions probably explains why organisms have many different genes encoding helicases. The multiplicity of helicase genes was not fully appreciated until the advent of whole-genome sequencing. For example, the yeast Saccharomyces cerevisiae has 134 open reading frames (ORFs), ~2% of its genome, with helicase-like features1. Because only a few of these genes have been tested for helicase activity by biochemical methods, the number of helicases is not known. It will be of interest to determine the specific roles of each putative helicase and also their extent of functional overlap. (See http://www.expasy.ch/linder/helicases_list.html for an updated analysis of Saccharomyces helicases.) This article concerns DNA helicases and focuses specifically on the recently discovered Pif1p subfamily of DNA helicases. Most DNA helicases require a single strand of substrate DNA for binding. DNA helicases that bind to 3′ single-strand DNA are said to unwind DNA in the 3′ to 5′ direction, whereas 5′ to 3′ helicases bind to 5′ single-strand DNA and unwind DNA in a 5′ to 3′ direction. Some helicases, such as the Escherichia coli RecBC helicase, can load onto duplex DNA. Nevertheless, the RecBC helicase translocates on only one strand of the DNA helix, such that it moves in a 3′ to 5′ direction2. During translocation on their DNA substrates, helicases are thought to contact mostly the phosphodiester backbone. Therefore, the

association of helicases with DNA is not generally thought to be sequence specific. Helicases are classified into superfamilies3, and most helicases are in superfamily I (SFI) and SFII. Members of SFI and SFII contain seven motifs. Although these motifs are spread throughout a ~300–500 amino acid region (herein called the helicase domain), structure analyses reveal that the motifs are clustered in the known crystal structures, and each is probably crucial for enzyme activity (reviewed in Ref. 4). Helicase subfamilies

Although the presence of the seven helicase motifs are diagnostic for SFI and SFII helicases, these motifs are short and quite degenerate (for consensus motifs, see Fig. 1, SFI; Fig. 2, SFII). Indeed, when analyzed by computer programs such as BLAST5 or FASTA6, their presence alone is not sufficient to confer significant sequence similarity on the two proteins containing them3. Helicase subfamilies are defined as a group of proteins that have highly significant sequence similarity by the criteria of computer searches. Although there are multiple helicase subfamilies, there is very little understanding of the functional significance underlying the amino acid similarity that defines a given subfamily. The simplest possibility is that members of the same subfamily have similar functions. Alternatively (or in addition), their similarity might reflect their ability to recognize a common structure (e.g. a Holliday junction) or a DNA sequence (e.g. telomeric DNA) or their interaction with a common protein cofactor (e.g. DNA polymerase). Interest in helicases has increased with the realization that several inherited human diseases are caused by mutations in genes encoding helicases. The helicase genes that have been found to cause disease when mutated in humans are members of a helicase subfamily for which there is also a Saccharomyces member7. Analysis of helicase functions in genetically tractable model organisms, such as Saccharomyces, might provide information that is relevant to the functions of these helicases in humans. For example, probably the best-studied helicase subfamily, the RecQ subfamily of SFII helicases, is conserved from bacteria to humans (Fig. 2). Humans express at least five different RecQ-like proteins; three are known to cause disease upon mutation (Fig. 2). Loss of the WRN helicase causes Werner’s syndrome8, a premature aging syndrome. Mutation of the BLM helicase causes Bloom’s syndrome9, a disease associated with a dramatic increase in cancer susceptibility. Mutation of the RECQL4 gene is thought to contribute to Rothmund–Thomson syndrome10, a disease also characterized by premature aging and cancer. Remarkably, mutation of the single Saccharomyces RecQ-like gene, SGS1, causes a collection of defects

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253 IFYTGSAGTGKSILLREMIKVLKGIYGREN--VAVTASTGLAACNIGGITIHSFAGIGLGKGDADKLYKKVRRSRKHLRRWENIGALVVDEISMLDAELL PIF1 249 VFYTGSAGTGKSVILQTIIRQLSSLYGKES--IAITASTGLAAVTIGGSTLHKWSGIGIGNKTIDQLVKKIQSQKDLLAAWRYTKVLIIDEISMVDGNLL RRM3 327 IFFTGSAGTGKSVLLRKIIEVLKSKYRKQSDRVAVTASTGLAACNIGGVTLHSFAGVGLARESVDLLVSKIKKNKKCVNRWLRTRVLIIDEVSMVDAELM S. pombe 263 VFFTGSAGTGKSVILRRIIEMLPAGN------TYITAATGVAASQIGGITLHAFCGFRYENSTPEQCLKQVLRQNHMVRQWKQCSHLIIDEISMIDRDFF C. elegans 221 VFFTGSAGTGKSFLLRRIISALPPDG------TVATASTGVAACLIGGTTLHAFAGIGGGDATMQRCLELASRP-ANAQTWRKCKRLIIDEISMVDGQFF D. m. ~200 IFFTGSAGTGKSYLLKRILGSLPPTG------TVATASTGVAACHIGGTTLHAFAGIGSGQAPLAQCVALAQRP-GVRQGWLNCQRLVIDEISMVEADLF H. sapiens ++xGxAGoGKS xx+xxxoo +++DExo Helicase consensus A P T IV III PIF1 DKLDFIARKIRKNHQPFGGIQLIFCGDFFQLPPVSKDP-------NRPTKFAFESKAWKEGVKMTIMLQKVFRQRGDVKFIDMLNRMRLGNIDDETEREF RRM3 DKLEQIARRIRKNDDPFGGIQLVLTGDFFQLPPVAKKDE------HNVVKFCFESEMWKRCIQKTILLTKVFRQQ-DNKLIDILNAIRYGELTVDIAKTI S. pombe DKLEEVARVIRKDSKPFGGIQLVLTGDFFQLPPVPEN--------GKESKFCFESQTWKSALDFTIGLTHVFRQK-DEEFVKMLNELRLGKLSDESVRKF C. elegans EALEYVARTVRNNDKPFGGIQLIITGDFFQLPPVSKD----------EPVFCFESEAWSRCIQKTIVLKNVKRQN-DNVFVKILNNVRVGKCDFKSADIL EKIEAVARHIRRNDRPFGGIQLILCGDFLQLPPVIKGDFGAAPTATPQQRFCFQSSAWETCIQCVYELKQVHRQS-DPEFVKILNHLRIGHVNDSITSRL D. m. DKLEAVARAVRQQNKPFGGIQLIICGDFLQLPPVTKG--------SQPPRFCFQSKSWKRCVPVTLELTKVWRQA-DQTFISLLQAVRLGRCSDEVTRQL H. sapiens xx+xooxR ++++GDxoQ

PIF1 RRM3 S. pombe C. elegans D. m. H. sapiens

KKLSRPLP-DDEIIPAELYSTRMEVERANNSRLSKLPGQVHIFNAIDGGALEDEELKERLLQNFLAPKELHLKVGAQVMMVKN--LDATLVNGSLGKVIE RNLNRDIDYADGIAPTELYATRREVELSNVKKLQSLPGDLYEFKAVDN---APERYQAILDSSLMVEKVVALKEDAQVMMLKNK-PDVELVNGSLGKVLF KVLNRTIEYEDGLLPTELFPTRYEVERSNDMRMQQINQNPVTFTAIDSGTVRDKEFRDRLLQGCMAPATLVLKVNAQVMLIKN--IDDQLVNGSLGKVIG KESSKNQFP-SSVIPTKLCTHSDDADRINSSSIETTQGDAKTFHAYD-----DESFDTHAKARTLAQKKLVLKVGAQVMLIKNIDVIKGLCNGSRGFVEK AATSKQKIEGNGILATQLCSHTNDANSINESKLENLDGDKILFKADDS----DASMTRTLDQQIQAPSQLYLKVNAQVMLLKNINISNGLVNGARGVVVR QATASHKVGRDGIVATRLCTHQDDVALTNERRLQELPGKVHRFEAMDS----NPELASTLDAQCPVSQLLQLKLGAQVMLVKNLSVSRGLVNGARGVVVG

PIF1 RRM3 S. pombe C. elegans D. m. H. sapiens

FMDPETYF----CYEALTNDPSMPPEKLETWAENPSKLKAAMEREQSDGEESAVASRKSSVKEGFAKSDIGEPVSPLDSSVFDFMKRVKTDDEVVLENIK FVTESLVVKMKEIYKIVDDEVVMDMRLVSR------VIGNPLLKESKE------------------------FRQDLNAR--------------PLARLE FIDDET-------YQMEKKDAEMQG-------------RNAFEYDSLD-------------------------ISPFD-----------------LPDVK FSENG----------------------------------------------------------------------------------------------MEKD-----------------------------------------------------------------------------------------------FEAEGR----------------------------------------------------------------------------------------------

PIF1 RRM3 S. pombe C. elegans D. m. H. sapiens

RKEQLMQTIHQNSAGKRRLPLVRFKASDMSTRMVLVEPEDWAIEDENEKPLVSRVQLPLMLAWSLSIHKSQGQTLPKVKVDLRRVFEKGQAYVALSRAVS 123 RLKILINYAVKISPHKEKFPYVRWTVGKNKYIHELMVPERFPIDIPRENVGLERTQIPLMLCWALSIHKAQGQTIQRLKVDLRRIFEAGQVYVALSRAVT 32 QKKYKLIAMRKASSTAIKWPLVRFKLPNGGERTIVVQRETWNIELPNGEVQASRSQIPLILAYAISIHKAQGQTLDRVKVDLGRVFEKGQAYVALSRATT 52 ------------------NPMIRFVS--QADASIEIRRSKFSVRIPGSDAPLIRRQLPLQLAWAISIHKSQGMTLDCAEISLERVFADGQAYVALSRARS 53 ------------------LPVVRFKN--NQEYVCKHER--WIIKTASG-NHITRRQVPLKLAWAFSIHKSQGLTLDCVEMSLSKVFEAGQAYVALSRAKS 74 -----------------GLPQVRFLC--GVTEVIHADR--WTVQATGG-QLLSRQQLPLQLAWAMSIHKSQGMTLDCVEISLGRVFASGQAYVALSRAAS 55 xxT+xxxQG+o+ooV VA+TR xoo S K I G S

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Fig. 1. Alignment of the helicase domain of the PIF1 subfamily of DNA helicases. This figure is an extension of work presented as supplementary material in Ref. 13. Sequences were collected with tBLASTn using default parameters. These sequences were aligned using Clustal W42 and Gene Doc [K.B. Nicholas and H.B.J. Nicholas (1997) Gene Doc: a tool for editing and annotating multiple sequence alignments; distributed by the authors] to highlight sequence similarities. Only the helicase domain of each protein is shown. Roman numerals indicate helicase motifs. Motif I is often referred to as the Walker A box and motif II as the Walker B box. An asterisk marks the residue in motif I whose mutation is discussed in the text. Numbers to the right of each helicase name indicate the number of amino acids from motif I to the N-terminus of the protein. The length of the human protein from motif I to the N-terminus is not known precisely but was estimated by matching ESTs in the database to a BAC (AC023671) that contains the hPIF gene. Numbers at the bottom right after motif VI indicate the number of amino acids to the C-terminus of the protein. Residues in red are 100% conserved, allowing for conservative amino acid changes. Yellow indicates 80% conservation and blue is 60% conservation. The helicase consensus sequence is from Ref. 3, as reviewed in Ref. 4. The amino acid consensus sequence indicates residues conserved in at least 75% of the SFI helicases known in 1993. Key: + indicates a hydrophobic residue, o indicates hydrophilic and × any residue. Dashes indicate gaps in the alignment. All eukaryotic Pif1p-like ORFs with e values of less than 10–40, except for an open reading frame (ORF) from Candida maltosa, are included. (The 365 amino acid C. maltosa sequence was not included because it is a partial sequence that lacks the region N-terminal of motif III.) In addition to the eukaryotic ORFs, the Chilo iridescent virus, a linear double-stranded virus that infects the rice stem borer insect, also encodes a Pif1-like protein (e value 10–48). The total length of each protein in amino acids is 859 (PIF1 Saccharomyces cerevisiae), 723 (RRM3 Saccharomyces cerevisiae), 805 (Schizosaccharomyces pombe), 677 (Caenorhabditis elegans), 663 [Drosophila melanogaster (D. m.)] and about 627 (Homo sapiens). The sequence for the helicase domain of the human Pif1 presented in Ref. 13 was extended through motif I using additional ESTs in the human database.

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that are argued to mimic the phenotypes of aged yeast cells11,12. The Saccharomyces Pif1p helicase inhibits telomerase

Work from our laboratory has documented a new SFI helicase subfamily, the Pif1 subfamily13 (Fig. 1). We encountered PIF1, the prototype member of this group, in a genetic screen designed to identify Saccharomyces genes whose mutation alters telomeres14. One such mutant affected not only telomeres but also the maintenance of mitochondrial DNA. When the wild-type copy of the mutant gene was cloned, it was found to be PIF1, which encodes a 5′ to 3′ DNA helicase15,16 that is important for maintenance and recombination of mitochondrial DNA17. Given the mitochondrial function of Pif1p, its effects on telomeres could have been a secondary consequence of the respiratory deficiency seen in its absence. However, there are point mutations in PIF1 that separate its mitochondrial and nuclear functions14. Strains with

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I RecQ E. coli SGS1 S. cerevisiae rqh1+S. pombe E03A3.2 C. elegans RecQ4 D. m. RecQL4 H. sapiens BLM H. sapiens WRN H. sapiens Helicase consensus

Ia

43 DCLVVMPTGGGKSLCYQIPALLLNG----LTVVVSPLISLMKDQVDQLQANGVAAACLNSTQTREQQLEVMTGCRTG--QIRLLYIAPERLMLDN----FLE 694 DVFVLMPTGGGKSLCYQLPAVVKSGKTHGTTIVISPLISLMQDQVEHLLNKNIKASMFSSRGTAEQRRQTFNLFING--LLDLVYISPEMISASEQCKRAIS 535 DVFILMPTGGGKSLCYQLPAVIEGGASRGVTLVISPLLSLMQDQLDHLRKLNIPSLPLSGEQPADERRQVISFLMAKNVLVKLLYVTPEGLASNGAITRVLK 195 SSLVTLSTGSGKSLCYQLPAYLYSRKVGAITLVISPLVSLMEDQVTGVP-HFLRAHCLHTNQTAPQRMKIQQMIANG--EIDILLVSPEAVVAGERATGFGA 837 DVYVSLPTGAGKSLCYQLPAVVHGG----ITVVISPLIALMKDQISSLKRKGIPCETLNSTLTTVERSRIMGELAKEKPTIRMLYLTAEGVATDGTKKLLNG 496 STLLVLPTGAGKSLCYQLPALLYSRRSPCLTLVVSPLLSLMDDQVSGLP-PCLKAACIHSGMTRKQRESVLQKIRAA--QVHVLMLTPEALVG----AGGLP 683 DCFILMPTGGGKSLCYQLPACVSPG----VTVVISPLRSLIVDQVQKLTSLDIPATYLTGDKTDSEATNIYLQLSKKDPIIKLLYVTPEKICASNRLISTLE 565 DNVAVMATGYGKSLCFQYPPVYVGK----IGLVISPLISLMEDQVLQLKMSNIPACFLGSAQSEN----VLTDIKLG--KYRIVYVTPEYCSGNMG--LLQQ ++xxxoGxGKT x+++xPoo S

II RecQ E. coli SGS1 S. cerevisiae rqh1+S. pombe E03A3.2 C. elegans RecQ4 D. m. RecQL4 H. sapiens BLM H. sapiens WRN H. sapiens

III

HLAHWN-PVLLAVDEAHCISQWGHDFRPEYAALG-QLRQRFPTLPFMALTATADDTTRQDIVRLLGLND--PLIQISSFDRPNIRYM------------LME RLYADGKLARIVVDEAHCVSNWGHDFRPDYKELK-FFKREYPDIPMIALTATASEQVRMDIIHNLELKE--PVFLKQSFNRTNLYYEVN------------SLYERKLLARIVIDEAHCVSHWGHDFRPDYKQLG-LLRDRYQGIPFMALTATANEIVKKDIINTLRMEN--CLELKSSFNRPNLFYEIK------------ILRQLPPIAFACIDEAHCVSQWSHNFRPSYLMICKVLRKNLGVRTVLGLTATATLPTRVSIINHLGISDGERGIISDIPLPDNLVLS--------------LANRDV-LRYIVVDEAHCVTQWGHDFRPDYLTLG-SLRDVCPGVPWVALTATANAKAQDDIAFQLKLRN--PESFKSGTYRDNLFYDNHMASFITKCLTVDA PAAQLPPVAFACIDEAHCLSQWSHNFRPCYLRVCKVLRERMGVHCFLGLTATATRRTASDVAQHLAVAE-EPDLHGPAPVPTNLHLS--------------NLYERKLLARFVIDEAHCVSQWGHDFRQDYKRMN-MLRQKFPSVPVMALTATANPRVQKDILTQLKILR--PQVFSMSFNRHNLKYYVLP-----------LEADIG-ITLIAVDEAHCISEWGHDFRDSFRKLG-SLKTALPMVPIVALTATASSSIREDIVRCLNLRN--PQITCTGFDRPNLYLE------------VRR +x+SATxxx +++DExH TGS

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IV RecQ E. coli SGS1 S. cerevisiae rqh1+S. pombe E03A3.2 C. elegans RecQ4 D. m. RecQL4 H. sapiens BLM H. sapiens WRN H. sapiens

KFKPLDQLMRYVQEQRG------KSGIIYCNSRAKVEDTAAALQSKGIS------------------AAAYHAGLENNVRADVQEKFQRDD-LQIVVATVAF ---KKTKNTIFEICDAVKSRFKNQTGIIYCHSKKSCEQTSAQMQRNGIK------------------CAYYHAGMEPDERLSVQKAWQADE-IQVICATVAF ----PKKDLYTELYRFISNGHLHESGIIYCLSRTSCEQVAAKLRNDYGLK-----------------AWHYHAGLEKVERQRIQNEWQSGS-YKIIVATIAF VSKDENRDAALLQLLNSERFEPCQSIIIYCTRRDECERIAGFIRTCVQDRREPTQDQTKKRKRVNWQAEPYHAGMPASRRRTVQKAFMSNE-LRIVVATIAF KTSSSNLTKHEKAERSQNKKTFTGSAIVYCRSRNECGQVAKMLEIAGIP------------------AMAYHAGLGKKDRNEVQEKWMNNE-IPVVAATVAF VSMDRDTDQALLTLLQGKRFQNLDSIIIYCNRREDTERIAALLRTCLHAAWVPGSGGRAPKT----TAEAYHAGMCSRERRRVQRAFMQGQ-LRVVVATVAF ---KKPKKVAFDCLEWIRKHHPYDSGIIYCLSRRECDTMADTLQRDGLA------------------ALAYHAGLSDSARDEVQQKWINQDGCQVICATIAF KTGNILQDLQPFLVKTSSHWEFEGPTIIYCPSRKMTQQVTGELRKLNLS------------------CGTYHAGMSFSTRKDIHHRFVRDE-IQCVIATIAF ++Fxxoxo +xTxxx Y S

VI GMGINKPNVRFVVHFDIPRNIESYYQETGRAGR RecQ E. coli SGS1 S. cerevisiae GMGIDKPDVRFVYHFTVPRTLEGYYQETGRAGR GMGVDKGDVRFVIHHSFPKSLEGYYQETGRAGR rqh1+S. pombe E03A3.2 C. elegans GMGINKPDIRAVIHYNMPRNFESYVQEIGRAGR RecQ4 D. m. GMGIDKPDVRAVIHWSPSQNLAGYYQEAGRAGR RecQL4 H. sapiens GMGLDRPDVRAVLHLGLPPSFESYVQAVGRAGR BLM H. sapiens GMGIDKPDVRFVIHASLPKSVEGYYQESGRAGR WRN H. sapiens GMGINKADIRQVIHYGAPKDMESYYQEIGRAGR xxG+o+xo+ QxxGRxxR

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Fig. 2. Alignment of the helicase domain of selected members of the RecQ subfamily of DNA helicases. Methods of analysis, symbols, and organization are the same as for Fig. 1. The prototype member of the RecQ subfamily is the Escherichia coli RecQ DNA helicase. Only a subset of the more than 20 RecQ-like proteins in the database is presented. Caenorhabditis elegans, Drosophila melanogaster (D. m.) and Homo sapiens have other RecQ-like proteins in addition to those shown here. The total length of each protein in amino acids is 610 (RecQ E. coli), 1447 (SGS1 Saccharomyces cerevisiae), 1328 (rqh1+ Schizosaccharomyces pombe), 809 (E03A3.2 C. elegans), 1530 (RecQ4 D. melanogaster), 1208 (RecQL4 H. sapiens), 1417 (BLM H. sapiens) and 1432 (WRN H. sapiens). Those RecQ-like helicases that have been examined biochemically have a 3′ to 5′ polarity.

the pif1-m2 allele have wild-type mitochondrial DNA but mutant telomeres, whereas pif1-m1 cells are defective in maintenance of mitochondrial DNA but have wild-type telomeres. As predicted by these genetic studies, there are two forms of Pif1p, a longer form that is destined for the nucleus, and a shorter form present in mitochondria13. Cells lacking the nuclear form of Pif1p have three telomere phenotypes14. Telomeres are longer and more heterogeneous in pif1∆ or pif1-m2 cells compared with wild-type telomeres. Because there are many genes that affect telomere length, this phenotype by itself is not particularly noteworthy. However, Pif1p also has a dramatic effect on the formation of new telomeres. In wild-type cells, new http://tcb.trends.com

telomeres are added only rarely to spontaneous or induced double-strand breaks; instead, these breaks are almost always healed by homologous recombination14,18,19. However, in pif1 cells, the rate of de novo telomere addition is increased as much as 600-fold. Moreover, although wild-type cells form new telomeres infrequently, when they do so, they almost always use sites that have nearby stretches of telomeric or telomere-like sequences14,19. By contrast, pif1 cells add new telomeres at sites that bear little resemblance to telomeric DNA14. Finally, overexpression of Pif1p causes telomere shortening13. Taken together, these data suggest that Pif1p inhibits telomere lengthening and increases the specificity of telomere addition. There are two pathways that can lengthen Saccharomyces telomeric tracts of C1–3A/TG1–3 DNA: telomerase and recombination20. Elongation by telomerase requires at least five genes21, including TLC1, which encodes the RNA subunit of telomerase. Lengthening of telomeric tracts by recombination requires both Rad52p (Ref. 20) and Rad50p (Ref. 22). As telomere lengthening occurs in

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rad52 pif1 cells but not in tlc1 pif1 cells, Pif1p must inhibit telomerase, not recombination13. Point mutations in the ATP-binding pocket motif of Pif1p that eliminate helicase activity in vitro (marked by the asterisk in Fig. 1) also cause telomere lengthening in vivo. Thus, the enzymatic function of Pif1p is required for inhibition of telomerase13. By the criterion of chromatin immunoprecipitation, Pif1p is associated with telomeric DNA in vivo13. Therefore, Pif1p probably affects the telomerase pathway directly, rather than, for example, by inhibiting transcription or translation of a telomerase subunit. Pif1p is the prototype member of a new helicase subfamily

At the time that Pif1p was found to affect telomeres, there were two genes in the database with high similarity to PIF1: a second Saccharomyces gene, now called RRM3, and a partial ORF from the yeast Candida maltosa. Once the sequence of the entire Saccharomyces genome was known, it became clear that, of the 133 other helicase-like ORFs in Saccharomyces, only RRM3 has similarity to PIF1 by the criterion of a BLAST search. Subsequently, PIF1-like genes were isolated from fission yeast (rph1+, for: ‘RRM3 PIF1 homologue’) and humans (hPIF)13. PIF1-like genes from Caenorhabditis elegans and Drosophila melanogaster entered the database as a result of genome-sequencing projects. In all pairwise combinations, the seven PIF1-like genes are greater than 30% identical over a ~400 amino acid region that contains all seven helicase motifs (Fig. 1).

...Pif1p is the prototype member of a helicase subfamily that is conserved from yeasts to humans For comparison, the RecQ subfamily members shown in Fig. 2 are ≥30% identical over the ~300 amino acid helicase domain region (Fig. 2). Over the helicase motifs themselves, allowing for conservative substitutions, the Pif1p subfamily members range in similarity from 37% (motif IV) to 100% (motifs I and II) (Fig. 1). Again, for comparison, the similarity of the helicase motifs among the RecQ subfamily members shown in Fig. 2 ranges from 54% (motif I) to 87% (motif V). Given their high identity to a demonstrated DNA helicase, Pif1p, over each helicase motif, the other members of the Pif1p subfamily are most probably DNA helicases. Thus, the Saccharomyces Pif1p is the prototype member of a helicase subfamily that is conserved from yeasts to humans, and the level of similarity within the Pif1 subfamily is similar to that found in other helicase subfamilies. The high similarity of the Pif1 subfamily members is not limited to the helicase motifs but, as http://tcb.trends.com

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with the RecQ subfamily (Fig. 2), extends to regions between the motifs (Figs 1 and 2). However, the sequence N-terminal to the helicase domain displays no detectable similarity in either subfamily. In addition, within the Pif1 subfamily, the regions between helicase motifs IV and V, and also the regions C-terminal to the helicase domain, vary in size and sequence (Fig. 1). The C-terminal region of a subset of the RecQ-like helicases contains a motif that is predicted to form an α-secondary structure that might play a role in DNA binding23. Although no common motif is found in the C-terminal region of the Pif1-like proteins, this region is much smaller in the Pif1 subfamily than in the RecQ subfamily (compare Figs 1 and 2). Nonconserved regions within helicases are predicted to be responsible for enzyme specificity, for example by dictating protein–protein interactions, substrate recognition, subcellular localization or additional enzymatic activities (reviewed in Ref. 4). Consistent with this possibility, the nonconserved N-terminal portions of the yeast RecQ-like Sgs1p helicase interacts with topoisomerases24–26, and the N-terminal portion of Pif1p interacts with Cac1p, the largest subunit of chromatin assembly factor I (Ref. 27). Also, the N-terminal region of the human WRN protein has a nuclease motif 28 that is not found in other RecQ subfamily members. The two Saccharomyces Pif1-like proteins affect the same DNA substrates but have different effects

The existence of two PIF1-like genes in Saccharomyces provides an opportunity to explore the functional significance of the sequence similarity that characterizes this subfamily. Like PIF1, RRM3 is not essential29, but deletion of RRM3 yields none of the telomere or mitochondrial phenotypes of pif1 cells. Moreover, deletion of RRM3 in a pif1 strain partially suppresses both the telomere and mitochondrial phenotypes that characterize pif1 cells, suggesting that Pif1p and Rrm3p have opposing effects on both substrates (V.P. Schulz and V.A. Zakian, unpublished). Opposing actions of Pif1p and Rrm3p have been well documented in ribosomal DNA (rDNA). Using either a marker-loss assay30 or generation of rDNA circles29, rrm3 cells display a large increase in recombination in the rDNA locus. By contrast, pif1 cells have fewer rDNA circles than wild-type cells, and deleting PIF1 from an rrm3 strain partially suppresses the increase in rDNA circles seen in rrm3 cells29. In addition, rDNA fragments are detected in rrm3 but not in wild-type or pif1 DNA. As circle formation but not rDNA breakage depends on the recombination protein Rad52p (Ref. 29), these data suggest that breakage occurs independently of recombination and probably generates the substrate for recombination-mediated circle formation. In bacteria, chromosome breakage occurs when replication forks stall (reviewed in Ref. 31). Thus, we

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Fig. 3. The pattern of rDNA replication in wild-type32,33 and mutant29 Saccharomyces cells. Yeast rDNA is organized in 9.1-kb repeat units that contain 35S and 5S coding sequences (line 1). Each repeat also contains a potential replication origin (ARS, solid black circle) but only a subset of these origins is active in a given S phase (line 2). Replication is initially bidirectional (line 2), but most leftward-moving forks stop (line 3) when they reach the replication fork barrier (RFB; red bar). Because the RFB is a polar block to fork progression, the rightward-moving fork continues unimpeded (lines 3–7; green arrows denote moving replication forks). At the end of replication, a moving and an arrested fork converge at the RFB (lines 9 and 10). In the absence of rrm3, the rightward-moving fork pauses at multiple sites throughout the rDNA (indicated by the red dotted lines). In addition, Rrm3p is needed for efficient separation of forks converged at the RFB. In the absence of Pif1p, the RFB is less efficient, such that the leftward-moving fork has a greater probability of moving past the RFB (dotted fork, line 3).

considered that the effects of Rrm3p and Pif1p on rDNA recombination might be a secondary consequence of alterations in rDNA replication. The rDNA in budding yeast32,33 has an unusual mode of replication (Fig. 3). Replication begins within the nontranscribed spacer in a subset of the repeats. Replication initially proceeds bidirectionally. However, when the leftward-moving fork encounters a cis-acting site – the replication fork barrier (RFB) – it stops. The RFB is a polar block to fork progression, affecting only leftward-moving forks. Thus, the rightward-moving fork proceeds past each RFB it encounters in the adjacent repeats. As a consequence of this replication mode, at the end of rDNA replication, rightward-moving forks converge on forks stalled at the RFB in a subset of repeats32,33. Cis-acting sites that impede replication fork progression in rDNA have also been described in multicellular organisms34–36. In the absence of Rrm3p, replication fork progression is stalled at multiple sites throughout the rDNA29 (Fig. 3). Rrm3p activity is also important to resolve forks converged at the RFB. By contrast, Pif1p plays a role in maintaining the RFB such that, in the absence of Pif1p, the RFB is less effective at halting leftward-moving forks (Fig. 3). Because chromatin immunoprecipitation shows that both Rrm3p and Pif1p are associated with rDNA in vivo, their effects on rDNA replication are probably direct. Again, the highly related Pif1p and Rrm3p http://tcb.trends.com

have opposing effects on the same DNA substrate: Pif1p inhibits fork progression in the rDNA by helping to maintain the RFB, whereas Rrm3p is needed for efficient fork progression throughout the rDNA. The effects of these proteins on replication fork progression can also explain their opposing effects on rDNA breakage and recombination29. It has been difficult to identify the helicases that are responsible for fork progression during replication of chromosomal DNA in eukaryotic organisms. In the absence of Rrm3p, DNA replication pauses at discrete sites in the rDNA, and each pause maps at or near a site that is bound by a non-nucleosomal protein complex (Fig. 3). For example, forks pause near origins of DNA replication and near sites of transcription initiation and termination29. Rrm3p affects rDNA replication catalytically because point mutations in an invariant lysine residue that eliminate helicase activity in other helicases, including Pif1p (Ref. 13), have the same effect on rDNA replication as complete loss of Rrm3p (Ref. 29). These data suggest that Rrm3p is a replicative helicase for rDNA. The MCM complex is required for fork progression during Saccharomyces chromosomal DNA replication37. A subset of the MCM proteins form a complex that has helicase activity in vitro38, although it is not known whether the helicase activity of the MCM complex is essential for fork progression. Nevertheless, these data suggest that the MCM complex is the replicative helicase for most chromosomal DNA sequences. How can the data on the role of the MCM proteins be reconciled with the evidence that Rrm3p is a replicative helicase for rDNA? Perhaps the MCM complex does not act in rDNA. Alternatively, Rrm3p might augment MCM activity by helping the replication fork to bypass protein–DNA complexes. Our work with the Saccharomyces members of the Pif1 subfamily shows that both Pif1p and Rrm3p affect the same three substrates – telomeric, mitochondrial and ribosomal DNAs – but have different functions at each of them. Neither protein seems to have a genome-wide role in DNA replication because the rates of chromosome loss and overall mitotic recombination are not increased in their absence14,30. These data suggest a model in which the sequence similarity shared by Pif1p and Rrm3p reflects their ability to recognize or be recruited to the same DNA targets, rather than their having similar functions. A corollary of this hypothesis is that substrate recognition by the Pif1 subfamily is likely to be determined by residues within the ~400 amino acid region of high identity (Fig. 1) rather than by the nonconserved N- or C-terminal regions. Perspectives

The proposal that the sequence similarity of Pif1 subfamily helicases reflects common substrates can be tested by examining the functions of Pif1p-like

Opinion

Acknowledgements Work in our laboratory is supported by grants from the NIH. We thank A. Ivessa and M. Mateyak for their comments on the manuscript and members of our laboratory for allowing us to cite their unpublished data.

TRENDS in Cell Biology Vol.11 No.2 February 2001

proteins from other organisms. In addition, RRM3 is not essential, even though Rrm3p is needed for efficient fork progression throughout the rDNA29 and also plays a role in sister-chromatid separation at telomeres (A.S. Ivessa and V.A. Zakian, unpublished). Because completion of chromosomal DNA replication is essential, there is likely to be at least one other helicase that, like Rrm3p, promotes replication fork progression at rDNA and at telomeres (it need not be the same helicase at both substrates). As RRM3 and PIF1 are the only Saccharomyces members of the Pif1 subfamily, this interpretation predicts that a helicase that is unrelated to Rrm3p must act in its place to perform similar functions. The 3′ to 5′ RecQ-like helicase Sgs1p is an appealing candidate for a protein that is redundant with Rrm3p, at the rDNA, telomeres, or both. Like rrm3 cells, a strain lacking Sgs1p has an increase in

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rDNA recombination24 in addition to an increase in rDNA circles12. However, unlike Pif1p and Rrm3p, which so far have been shown to affect only a subset of the genome, Sgs1p probably has a more global role in DNA replication39,40. Increased recombination is a hallmark of cells lacking RecQ-like helicases from bacteria to yeast to humans. If Sgs1p is indeed partially redundant with Rrm3p, the generalized increase in recombination that accompanies loss of RecQ-like helicases might be, as with Rrm3p (Ref. 29), a secondary consequence of defects in DNA replication (reviewed in Ref. 41). If the human Pif1-like protein, like its Saccharomyces counterparts, is involved in maintenance of telomeric, ribosomal or mitochondrial DNA, it would not be surprising if its mutation, like that of the human RecQ-like proteins, affects aging, cancer, or both.

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