Virginia A. Zakian
FocusDNA replication and chromosome structure in yeast; telomeres; replication fork progression
My lab uses a combination of genetic and biochemical methods to study (1) telomere structure and replication and (2) replication fork progression. We work mainly in budding and fission yeasts with occasional forays into mammalian systems. Major accomplishments include:
Ciliate termini and YACs: We were the first to construct and characterize a linear artificial chromosome (YAC) (Dani and Zakian 1983 PNAS). In this and subsequent work (Pluta et al., 1984 PNAS), we were one of two groups to use ciliate telomeres to generate linear yeast episomes, a strategy that began the molecular era of yeast telomere biology.
Single strand telomere binding proteins: We isolated and characterized the first single-strand, sequence specific telomere DNA binding protein, the prototype of Pot1, from ciliates (Gottschling and Zakian 1986 Cell). In vitro, the heterodimeric complex is sufficient to distinguish authentic telomeres from DNA ends and to protect them from degradation. We were one of two groups to demonstrate that Cdc13 is the yeast functional equivalent of the ciliate G-strand binding protein (Lin and Zakian 1996 PNAS) and the first to show that it is telomere associated in vivo (Bourns et al., 1998 Mol. Cell. Biol; Tsukamoto 2001 Current Biol). Using genetic and biochemical approaches, we demonstrated that Cdc13 and the telomerase subunit Est1 interact directly, an interaction that is sufficient to bring telomerase to DNA ends in vitro (Qi and Zakian 2000 Genes and Develop.; Wu and Zakian 2011 PNAS). This association is cell cycle regulated, occurring in late S/G2 phase and defines one of the two pathways for recruiting telomerase to telomeres (Taggart et al., 2002 Science). In fission yeast the shelterin-like protein Ccq1 has a Cdc13-like function in recruiting telomerase, again via interaction with Est1 (Webb and Zakian 2012 Genes & Develop.).
Duplex telomere binding proteins: My lab was the first to show that the duplex sequence specific Rap1 protein binds telomeres in vivo where it affects telomere structure and chromosome stability (Conrad et al., 1990 Cell). These studies were the first chromatin immunoprecipitation (ChIP) of a yeast protein, a method we pioneered to study protein dynamics at yeast telomeres. We extended this work by showing that yeast telomeric DNA is assembled into a non-nucleosomal chromatin structure (Wright et al., 1992 Genes & Develop.).
Telomere position effects: We discovered telomere position effect, TPE, the transcriptional repression of genes near telomeres in budding yeast (Gottschling et al., 1990 Cell). We showed that telomeric silencing is due to proximity to telomeric sequence; i.e., it does not require proximity to a telomere (Stavenhagen and Zakian 1994 Genes & Develop.). We showed that telomeres also exert position effects on recombination (Stavenhagen and Zakian 1998 Genes & Develop.). Yeast TPE requires the ability of the telomere to fold back onto subtelomeric DNA (de Bruin et al., 2000 Mol. Cell. Biol). The Zakian lab was the first to use the LacO-GFP system to localize individual telomeres within the nucleus. Although yeast telomeres are often at the nuclear periphery, this localization does not require key silencing proteins, and localization and TPE are not obligatorily linked (Tham et al. 2001 Molecular Cell).
Telomere structure: By sequencing individual telomeres, we showed that only the terminal half of yeast telomeres is subject to lengthening and degradation (Wang and Zakian 1990 Mol Cell Biol). We made the surprising discovery that C-strand degradation occurs as a cell cycle regulated step in telomere biology that allows G-tails to be generated at the ends of linear chromosomes (Wellinger et al, 1993 Cell; Wellinger et al., 1996 Cell). This degradation occurs after conventional semi-conservative telomere replication, while C-strand re- synthesis occurs prior to mitosis (Wellinger et al. 1993 Mol. Cell. Biol.). In addition to interacting with Est1, Cdc13 also interacts with the catalytic subunit of DNA polymerase alpha (Qi and Zakian 2000 Genes and Develop.).
When this interaction is reduced, telomerase is more likely to lengthen telomeres. These data suggest that Cdc13 acts as a switch between promoting telomerase lengthening versus C-strand re-synthesis. By eliminating a single telomere in a controlled manner, we demonstrated that while telomeres are essential for the long-term stability of yeast chromosomes, a chromosome without a telomere can replicate and segregate for many generations before it is lost (Sandell and Zakian 1993 Cell). As part of this analysis, we discovered that even a single double strand break generates a robust checkpoint response, but that cells can ultimately adapt to a single break and resume cell division, even if the break is not repaired.
Telomere recombination: In many organisms, including yeast and human cells, recombination can maintain telomeres in cells lacking telomerase. We provided the first evidence that eukaryotic telomeres can be lengthened by gene conversion (Pluta and Zakian 1989 Nature; Wang and Zakian 1990 Nature). We also determined the structure of telomeres maintained by type II recombination and showed that they are generated by abrupt but relatively infrequent Rad50 dependent, Rif1 inhibited telomere lengthening (Teng and Zakian 1999 Mol Cell Biol; Teng et al., 2000 Molecular Cell). Type II recombination in yeast is remarkably similar to ALT in mammalian cells.
Cell cycle regulation of budding yeast telomerase: The Zakian lab pioneered the use of ChIP to determine mechanisms of telomerase recruitment as a function of both the cell cycle and telomere length. Although yeast telomerase is not active in G1 phase, the catalytic core of telomerase (Est2 and TLC1, telomerase RNA) are telomere associated at this time, although the complex is not engaged with the end of the chromosome (Taggart et al., 2002 Science; Sabourin et al. 2007 Molecular Cell). Est2/Tlc1 binding in G1 phase depends on a specific interaction between Yku80 and TLC1 RNA (Fisher et al., 2004 Nature Struct Mol Bio). This association defines one of two pathways, each of which is sufficient to maintain telomeric DNA via telomerase. The second pathway occurs in late S/G2 phase via interaction between Cdc13 and Est1 (Taggart et al., 2002 Science). As Est1 interacts directly with Est3 and Est1 is essential for Est3 to bind telomeres, the Cd13-Est1 association, also brings Est3 to telomeres (Tuzon et al. 2011 PLoS Genetics). As the proteins encoded by the telomerase minus alleles cdc13-2 and est1-60 are not defective in their interaction with each other (Wu and Zakian 2011 PNAS), their telomerase deficiency is not due to a recruitment defect. Rather, the Ming lab (Chen et al. 2018 Cell) demonstrated that Cdc13-2 and Est1-60 proteins are both deficient in telomerase activation.
Telomere length regulation of budding yeast telomerase: Telomerase binds preferentially to short telomeres in late S phase, which can explain their preferential lengthening by telomerase (Sabourin et al, 2007 Moleular Cell). Short telomeres have less Rif2 per telomere than wild type length telomeres, and this differential is required for preferential binding of telomerase to short telomeres (Sabourin et al. 2007 Molecular Cell, McGee et al. 2010 Nature Struct. Mol. Bio). Short telomeres also have elevated binding of the Mre11 complex, which recruits Tel1 checkpoint kinase preferentially to short telomeres, which then recruits telomerase (Goudsouzian et al 2006 Molecular Cell; Sabourin et al., 2007 Molecular Cell; McGee et al., 2010 Nature Struct. Mol. Bio). The Pif1 DNA helicase helps to channel telomerase to short telomeres by removing telomerase preferentially from longer telomeres (Phillips et al., 2015 PLoS Genetics).
Identifying new regulators of budding yeast telomerase. Genetic approaches identified ~400 genes that affect telomere length, although the telomere functions of most of these are still unknown. We used mass spectrometry (MS) to identify novel proteins that associate with the catalytic core of telomerase (Lin et al.
Nature Communications 2015). At the start of this project, only ten proteins were known subunits of telomerase, and all of them co-purified with telomerase in our MS study. An additional ~70 proteins associated with high stringency to telomerase in a DNase-independent manner. So far, we have characterized two sets of proteins that are subunits of known multi-protein complexes but had not been known previously to affect telomerase. First, we studied three subunits of the essential and highly conserved Cdc48-Npl4-Ufd1 complex that targets ubiquitinated proteins for degradation. Est1, a telomerase activator, is ~40 times more abundant in cells with reduced Cdc48, yet, paradoxically, telomeres are shorter. Furthermore, Est1 is mono-ubiquitinated and its cell cycle-regulated abundance is lost in Cdc48-deficient cells. Ufd4, an E3 ligase that was also discovered in our MS studies, ubiquinates Est1. Thus, in concert with Ufd4, the Cdc48 complex regulates telomerase by controlling the level and activity of Est1. More recently, we have concentrated on the telomerase-associated Pop1, Pop6 and Pop7 proteins (Lin et al. Nature Communications 2015), which are among the ~10 essential protein subunits of two highly conserved multi-protein-RNA complexes, RNase P and RNase MRP. The three Pop proteins bind TLC1, telomerase RNA. When cells are limited for any one of the three Pop proteins, telomeres are short yet paradoxically TLC1 levels are ~5 times higher than in WT cells. However, the TLC1 RNA that accumulates in pop mutant cells is defective, unable to assemble properly into a holoenzyme, which impairs the holoenzyme’s telomere binding. These defects can be explained by misfolding of TLC1. This misfolding is concentrated in three specific regions of TLC1, the Est1 binding site, the Est2 (catalytic subunit) binding site, and the template region of the RNA (Garcia et al., in preparation). Folding of other regions, such as the Yku80 and Sm ring binding sites, are not Pop dependent. Work from other labs suggests that the role of Pop proteins in regulating telomerase RNA is highly conserved.
Regulation of S. pombe telomerase. We used S. pombe to determine if the mechanisms of telomerase regulation and/or fork progression found in budding yeast are similar in these two distantly related organisms (see later sections for summary of fork progression studies in S. pombe). To study telomerase regulation, we started with isolation and characterization of Ter1, telomerase RNA, whose identity had eluded the field despite the availability of the sequence of the entire S. pombe genome (Webb and Zakian 2008 Nature Struc. Mol. Bio). We found that Ccq1, a subunit of shelterin, interacts with Est1, an interaction that defines one of the two essential pathways for telomerase recruitment; the same region in Est1 also interacts with Ter1, which promotes and stabilizes telomerase-telomere associations (Webb CJ and Zakian VA. 2012 Genes & Develop.).
These interactions are reminiscent of those between budding yeast Est1 with both Cdc13 and TLC1 RNA. We discovered that Ter1, like human telomerase RNA, contains a stem terminus element (STE) that is required for telomerase action (Webb and Zakian 2015 PNAS). By isolating a ter1 allele with a partially defective STE, we found that the mutant STE altered the sequence of telomeric DNA by impairing the function of the template boundary element, even though in the 2D structure of Ter1 RNA, these two functional elements are far apart. The changes in telomeric sequence resulted in an improperly assembled shelterin and hence short telomeres. More recently, we used fission yeast as a model to determine mechanisms of environmental stress-induced telomere shortening (Pohl et al., under review). In response to growth at higher temperatures that still support a near WT growth rate, telomeres rapidly (and reversibly) shortened, with the degree of shortening related to the level of heat stress. The stress response was complex and multi-faceted with changes in the abundance of two telomerase subunits and three shelterin components. All of these changes occurred by post-transcriptional mechanisms. One of the key changes in response to heat was reduced telomere bound Tpz1, a reduction that promotes telomere maintenance and cell survival in response to heat.
The Pif1 DNA helicase inhibits telomerase: The S. cerevisiae Pif1 DNA helicase uses its helicase activity to suppress telomerase-mediated telomere elongation and de novo telomere addition to double strand breaks (Schulz and Zakian 1994 Cell; Zhou et al., 2000 Science). By both in vivo and in vitro assays, Pif1 uses its ATPase activity to evict telomerase from DNA ends (Boulé et al., 2005 Nature). Using both ensemble and single molecule biochemistry, we showed that Pif1 has the unusual property of preferentially displacing RNA from DNA, suggesting that it dislodges telomerase by unwinding the telomerase RNA/telomeric DNA hybrid (Boulé and Zakian 2005 NAR; Zhou et al., 2014 eLife). We discovered that a second yeast helicase, Hrq1, a homolog of human RecQ4 whose mutation causes increased susceptibility to cancer and premature aging, acts non- catalytically to inhibit telomerase at both telomeres and DNA breaks. Hrq1 is also required for repair of DNA inter-strand cross links (Bochman et al., 2014 Cell Reports).
Rrm3 and Pfh1 DNA helicases promote fork progression through stable protein complexes: Largely from work in my lab, Pif1 family helicases were established as the first example of eukaryotic helicases that affect fork progression at diverse, naturally occurring sites that imped fork progression in every S phase. Virtually all eukaryotes and many bacteria encode Pif1 family DNA helicases (Zhou et al, 2000 Science). Some, like budding yeast, encode two (Pif1 and Rrm3) while most organisms, like fission yeast (Pfh1) and humans (hPIF1) encode only one. Pif1 family helicases have remarkably diverse functions, any one of which could explain why mutation of human PIF1 is associated with increased breast cancer risk (Chisholm et al. 2012 PLoS One). Rrm3 promotes fork progression at over 1000 genomic loci, including multiple sites within the ribosomal DNA (rDNA) (Ivessa et al., 2000 Cell), telomeres (Ivessa et al., 2002 Genes & Develop.), polymerase III transcribed genes, centromeres, silencers, and converged replication forks (Ivessa et al., 2003 Molecular Cell; Azvolinsky et al., 2009 Molecular Cell; Fachinetti et al., 2010 Molecular Cell). Rrm3-sensitive sites are assembled into stable protein complexes, and it is these complexes that make their replication Rrm3-dependent (Ivessa et al., 2003 Molecular Cell; Torres et al., 2004 Genes & Develop.). At some of these sites (e.g., tRNA genes, centromeres), Pif1 is a backup for Rrm3 in replication through these complexes (Tran et al., 2017 Nature Communications; Chen et al., in prep.). In vivo, Pif1 family helicases, suppress R-loop mediated DNA damage at tRNA genes (Tran et al. 2016 Nature Communications) and centromeres (Chen, et al., in prep). Fission yeast Pfh1 has many of the same in vivo and in vitro properties as the two budding yeast helicases, except that it does not inhibit telomerase (Pinter et al,. 2008 Molec. Cell. Biol.; Sabouri et al., 2012 Genes & Dev; McDonald et al., 2014 DNA Repair; Sabouri et al., 2015 BMC Biology; McDonald et al., 2016 PLoS Genetics). Human PIF1, like its budding and fission yeast homologs, affects maintenance of telomeres and mitochondrial DNA (Follonier and Zakian, in preparation). During analysis of the impact of Rrm3 on fork progression, we discovered that highly transcribed genes impede fork progression in an orientation independent method (Azvolinky et al., 2009 Molecular Cell), the first demonstration of what is now known to be a general phenomenon in eukaryotes, including in S. pombe (Sabouri et al., 2012 Genes & Develop; McDonald et al., 2016.). In another fork progression study, we found that tri-nucleotide repeats cause replication fork slowing and DNA breakage in a length dependent manner in budding yeast and obtained the first non-clinical expansions of trinucleotide repeats of the size associated with human disease (Freudenreich et al. 1998 Science).
Pif1 family helicases from bacteria to humans efficiently unwind G-quadruplex (G4) DNA: G4 DNA is an extremely stable DNA secondary structure held together by multiple G-G base pairs. In both budding and fission yeasts, G4 motifs (sequences that can form G4 structures in vitro) are among the preferred binding sites for budding yeast Pif1 and fission yeast Pfh1 (Pasechke et al., 2011 Cell; Sabouri et al. 2014 BMC Biology). In the absence of Pif1 or Pfh1, DNA replication slows and forks often break at G4 motifs that are Pif1/Pfh1-associated in wild type cells. By both ensemble and single molecule biochemistry, budding yeast and bacterial Pif1 helicases efficiently unwind G4 DNA (Paeschke et al., 2013 Nature; Zhou et al., 2014 eLife). Bacterial, S. pombe, and human Pif1 enzymes can all suppress G4-associated DNA damage in S. cerevisiae (Paeschke et al., 2013 Nature). Currently, we are extending this work using genome-wide approaches to determine how structural features of individual G4 motifs, as well as the impact of G4-stabilizing drugs, affect the efficiency of G4-mediated fork slowing and breakage (Chen, Pott, Zakian, in prep).
References from Zakian lab that are cited in this summary:
Azvolinsky A, Giresi PG, Lieb JD, Zakian VA. (2009) Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae. Molecular Cell 34: 722-734 (featured article). PMC2728070 (must read Faculty1000)
Bochman* ML, Paeschke K*, Chan A, and Zakian VA (2014) Hrq1, a homolog of the human RecQ4 helicase, acts catalytically and structurally to promote genome integrity (*co-first authors). Cell Reports. 6: 346-356. PMC3933191
Boulé JB and Zakian VA. (2007). The yeast Pif1p DNA helicase preferentially unwinds RNA DNA substrates. Nucleic Acids Res. 35: 5809-5818. PMC2034482
Boulé JB, Vega LR, and Zakian VA. (2005) The yeast Pif1p helicase removes telomerase from telomeric DNA. Nature
438: 57-61. (Faculty 1000 selected paper)
Bourns BB, Alexander MK, Smith AM, and Zakian VA. (1998) Sir proteins, Rif proteins, and Cdc13p bind Saccharomyces telomeres in vivo. Mol. Cell. Biol. 18: 5600-5608.
Chan A, Boule JB, and Zakian VA. (2008) Two pathways recruit telomerase to S. cerevisiae telomeres. PLoS Genet. 4: e1000236. PMC2567097
Chisholm KM, Aubert SD, Freese KP, Zakian VA, King M-C, and Welcsh PL. (2012) A genomewide screen for suppressors of Alu-mediated rearrangements reveals a role for PIF1. PLoS One. 7: e30748. PMC3276492
Conrad MN, Wright J, Wolf A and Zakian VA. (1990) RAP1 protein interacts with yeast telomeres in vivo: Overproduction alters telomere structure and decreases chromosome stability. Cell 63: 739-750.
Dani GM and Zakian VA. (1983) Mitotic and meiotic stability of linear plasmids in yeast. Proc. Natl. Acad. Sci. USA 80: 3406‑ 3410.
de Bruin D, Kantrow SM, Liberatore RA, and Zakian VA. (2000) Telomere folding is required for the stable maintenance of telomere position effects in yeast. Mol. Cell. Biol. 20: 7991-8000. PMC86409
Fachinetti D, Bermejo R, Cocito A, Minardi S, Katou Y, Kanoh Y, Shirahige K, Azvolinsky A, Zakian VA, and Foiani M. (2010) Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements. Molecular Cell. 39: 595-605. (issue highlight) PMC3041477
Fisher TS, Taggart AKP, and Zakian VA. (2004) Cell cycle-dependent regulation of yeast telomerase by Ku. Nature Struct. Mol. Biol. 11: 1198-1205.
Freudenreich CH, Kantrow SM, and Zakian VA. (1998) Expansion and length-dependent fragility of CTG repeats in yeast. Science 279: 853-856.
Gottschling DE and Zakian VA. (1986) Telomere Proteins: Specific recognition and protection of natural termini of Oxytricha macronuclear DNA. Cell 47: 195 205.
Gottschling DE, Aparicio DM, Billington BL and Zakian VA. (1990) Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63: 751-762.
Goudsouzian LK, Tuzon CT and Zakian VA. (2006) S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association. Molecular Cell. 24: 603-610. PMID: 17188035
Ivessa AS, Zhou J-Q, and Zakian VA. (2000) The Saccharomyces Pif1p DNA helicase and the highly related Rrm3p have opposite effects on replication fork progression in ribosomal DNA. Cell. 100: 479-489.
Ivessa AS, Zhou J-Q, Schulz VP, Monson EK, and Zakian VA. (2002) Saccharomyces Rrm3p, a 5’ to 3’ DNA helicase that promotes replication fork progression through telomeric and sub-telomeric DNA. Genes & Develop. 16: 1383-1396. PMC186315
Ivessa AS, Lenzmeier BA, Bessler JB, Goudsouzian LK, Schnakenberg SL and Zakian VA. (2003) The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past non-histone protein-DNA complexes. Molecular Cell. 12: 1525-1536. (Faculty 1000 selected paper) PMID:14690605
Lin JJ and Zakian VA. (1996) The Saccharomyces CDC13 protein is a single-strand TG1-3 telomeric DNA binding protein in vitro that affects telomere behavior in vivo. Proc. Natl. Acad. Sci. USA 93: 13760-13765.
Lin, KW, McDonald, KR, Guise, AJ, Chan, A, Cristea, IM, and Zakian, VA (2015) Proteomics of budding yeast telomerase: the telomerase associated Cdc48-Npl4-Ufd1 complex regulates Est1 abundance and telomere length. Nature Communications. 6:8290. doi: 10.1038/ncomms9290. PMID: 26365526).
*McDonald, KR, *Sabouri, N, *Webb, CJ, and Zakian, VA (2014) Pfh1 is a positive regulator of telomere replication and telomere length. DNA Repair (Amst). 2014 Oct 7. pii: S1568-7864(14)00239-0. doi: 10.1016/j.dnarep.2014.09.008.
McDonald KR, Guise AJ, Pourbozorgi-Langroudi PCristea IM, Zakian VA , Capra JA, and Sabouri N (2016) Pfh1 is an accessory replicative helicase that interacts with the replisome to facilitate fork progression and preserve genome integrity. PLoS Genetics. 12: e1006238. PMID:27185885 doi:10.1371/journal.pgen.1006238
McGee JS, Phillips JA, Chan A, Sabourin M, Paeschke K and Zakian VA. (2010) Reduced Rif2 and lack of Mec1 target short telomeres for elongation rather than double-strand break repair. Nature Struct. Molec. Biol. 17: 1438-1445.
PMC3058685 (Faculty 1000 selected paper)
Paeschke K, Bochman ML, Cejka P, Friedman KL, Kowalczykowski SC, and Zakian VA. (2013) Pif1 helicases from bacteria to humans suppress genome instability at G-quadruplex DNA motifs. Nature 497:458-62.; full length article; news and views, Mirkin, S. 2013. DNA replication: driving past four-stranded snags. Nature 497(7450):449-50.; commentary in Princeton Journal Watch; Science 360 News watch).
Paeschke K, Capra JA, and Zakian VA. (2011) DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase. Cell 145: 678-691. (highlighted in Nat. Struc. Mol. Biol; Nat. Rev. Mol. Cell Biol. PMC3129610
Phillips JA, Chan, A, Paeschke K*, Zakian VA*. (2015) The Pif1 DNA helicase inhibits the frequency and processivity of S. cerevisiae telomerase action and helps target telomerase to short telomeres. PLoS Genetics DOI: 10.1371/journal.pgen.1005186. PMC4408051
Pluta AF and Zakian VA. (1989) Recombination occurs during telomere formation in yeast. Nature 337: 429-433.
Pluta AF, Dani GM, Spear BB and Zakian VA. (1984) Elaboration of telomeres in yeast: Recognition and modification of termini from Oxytricha macronuclear DNA. Proc. Natl. Acad. Sci. USA 81: 1475‑ 1479.
Pohl TJ, CJ Webb, Y Wu, VA Zakian (under review) Adaptive telomere shortening in response to environmental stress requires shelterin rearrangement
Qi H and Zakian VA. (2000) The Saccharomyces telomere binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase a and the telomerase associated Est1 protein. Genes & Develop. 14: 1777-1788.
Runge K and Zakian VA. (1989) Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation. Mol. Cell. Biol. 9: 1488-1497.
Sabouri N, McDonald K, Webb CJ, Cristea I, and Zakian VA. (2012) DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase. Genes & Develop. 26: 581-593. PMC3315119 (commentary in Shimada K, Gasser SM. 2012 Curr. Biol. 22: R404-5 DNA replication: pif1 pulls the plug on stalled replication forks.)
*Sabouri, N, *Capra JA, and Zakian VA (2014) The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage (co-first authors). BMC Biology 12:101. PMID:25471935
Sabourin M, Tuzon, CT, and Zakian VA. (2007) Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae. Molecular Cell 27: 550-561. (immediate early publication article) PMC2650483
Sandell LL and Zakian VA. (1993) Loss of a yeast telomere: arrest, recovery and chromosome loss. Cell 75: 729-739.
Schulz VP and Zakian VA. (1994) The Saccharomyces PlFl DNA helicase inhibits telomere elongation and de novo telomere formation Cell 76: 145-155.
Stavenhagen J and Zakian VA. (1994) Internal tracts of telomeric DNA act as silencers in Saccharomyces cerevisiae.
Genes & Develop. 8: 1411-1422.
Stavenhagen JB and Zakian VA. (1998) Yeast telomeres exert a position effect on recombination between internal tracts of yeast telomeric DNA. Genes & Develop. 12: 3044-3058.
Taggart AKP, Teng S-C, and Zakian VA. (2002) Est1p as a cell cycle regulated activator of telomere-bound telomerase.
Science. 297: 1023-1026. (Faculty 1000 selected paper).
Teng SC and Zakian VA. (1999) Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol. Cell. Biol. 19: 8083-8093.
Teng S-C, Chang J, McCowan B and Zakian VA. (2000) Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process. Molecular Cell. 6: 947-952. PMID:11090632
Tham W-H, Wyithe JSB, Ko Ferrigno P, Silver PA and Zakian VA. (2001) Localization of yeast telomeres to the nuclear periphery is separable from transcriptional repression and telomere stability functions. Molecular Cell. 8: 189-199. PMID: 11511372
Torres JZ*, Bessler JB*, and Zakian VA. (2004) Local chromatin structure at the ribosomal DNA causes replication fork pausing and genome instability in the absence of the S. cerevisiae DNA helicase Rrm3p. Genes & Develop. 18: 498-503 (*co- first authors) PMC374232
Tran, P L T, Chen, C-F, Pohl, TJ, Pott, S, Chan, A, and Zakian VA Chi-Fu (2017) PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes. Nature Communications. 8:15025 doi: 10.1038/ncomms15025
Tsukamoto Y, Taggart AKP and Zakian VA. (2001) The role of the Mre11-Rad50-Xrs2 complex in telomerase mediated lengthening of Saccharomyces telomeres. Current Biology. 11: 1328-1335.
Tuzon CT*, Wu Y*, Chan A, and Zakian VA. (2011) The S. cerevisiae telomerase subunit Est3 binds telomeres in a cell cycle and Est1 dependent manner and interacts directly with Est1 in vitro (*co-first authors) PLoS Genetics. 7: e1002060. PMC3088721
Wang S-S and Zakian VA. (1990a) Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains. Mol. Cell. Biol. 10: 4415-4419.
Wang S-S and Zakian VA. (1990b) Telomere-telomere recombination provides an express pathway for telomere acquisition. Nature 345: 456-458.
Webb CJ and Zakian VA. (2008) Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA. Nature Struct. Molec. Biol. 15: 34-42. (news and views in same issue) PMC2703720
Webb CJ and Zakian VA. (2012). Schizosaccharomyces pombe Ccq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase-telomere association. Genes & Develop. 26: 82-91. PMC3258969
Webb CJ and Zakian VA (2015) Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding. Proc. Natl. Acad. Sci. USA 112: 1312–11317, doi: 10.1073/pnas.1503157112
Wellinger RJ, Wolf A and Zakian VA. (1993) Saccharomyces telomeres acquire single-strand TG1-3 tails late in S phase.Cell 72: 51-60.
Wellinger RJ, Wolf AJ, and Zakian VA. (1993) Origin activation and formation of single-strand TG1-3 tails occur sequentially in late S phase on a linear plasmid. Mol. Cell. Biol. 13:4057-4065.
Wellinger RJ, Etier K, Labreque P and Zakian VA. (1996) Evidence for a new step in telomere maintenance. Cell 85: 423-433.
Wright JH, Gottschling DE and Zakian VA. (1992) Saccharomyces telomeres assume a non-nucleosomal chromatinstructure. Genes & Develop. 6: 197-210.
Wu Y and Zakian VA. (2011) The telomeric Cdc13 protein interacts directly with the telomerase subunit Est1 to bring it to telomeric DNA ends in vitro. Proc. Natl. Acad. Sci. USA 108: 20362-20369. PMC3251085
Zhou R, ZhangJ, Bochman, ML, Zakian VA and Ha TJ (2014) Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA. eLIFE. 3:e02190. PMID: 24843019 (commentary: Chistol G and Walter J (2014) Molecular watchdogs on genome patrol. eLIFE. 3:e02854.)
Zhou J-Q, Monson EK, Teng S-C, Schulz VP, and Zakian VA. (2000) Pif1p helicase, a catalytic inhibitor of telomerase in yeast. Science. 289: 771-774.
Virginia A. Zakian is the Harry C. Weiss Professor in the Life Sciences in the Department of Molecular Biology at Princeton University. She received her B.S. in Biology from Cornell University and her Ph.D. in Biology in 1975 for research carried out in the lab of Dr. Joseph G. Gall at Yale University. In 1979, after three years of post doctoral work (first at Princeton University with Dr. Arnold J. Levine in Biochemistry and then the University of Washington with Dr.Walton F. Fangman in Genetics), she started her own lab at the Fred Hutchinson Cancer Research Center in Seattle WA. She became a tenured full professor in 1987. In 1995, she joined the faculty of the Department of Molecular Biology at Princeton University where she is currently the Harry C. Wiess Professor in the Life Sciences where her research concerns chromosome structure and replication, with a focus on telomeres, the ends of chromosomes and replication fork progression through natural replication barriers.
Virginia Zakian has made critical contributions in two areas of chromosome biology: telomeres and replication fork progression. Her lab used ciliates to isolate the first telomere single-strand DNA binding proteins, the prototype of Pot1, and demonstrated that they protect DNA ends from degradation. Her lab discovered telomeric silencing and cell cycle dependent degradation of C-strand telomeric DNA in budding yeast, features now known to occur in diverse eukaryotes. They also identified proteins required for cell cycle and length-dependent regulation of telomerase. Her lab discovered that components of two different highly conserved multi-protein complexes regulate the abundance and activity of a telomerase subunit: the Cdc48 complex regulates Est1 and three subunits of the RNaseP/MRP complexes regulate telomerase RNA. Her work on fork progression focuses on the Pif1 family of DNA helicases, which her lab showed are conserved from bacteria to humans. These studies began with the discovery that the budding yeast Pif1 acts catalytically to eject telomerase from telomeres and double strand breaks, thereby inhibiting telomerase. Two other members of the Pif1 helicase family, budding yeast Rrm3 and fission yeast Pfh1, promote semi-conservative replication through telomeric DNA. Moreover, budding and fission yeast Pif1 family helicases have more general roles in suppressing the replication stress that arises at naturally occurring replication impediments, such as stable protein complexes, converged replication forks, and DNA secondary structures. Her lab also discovered that highly transcribed RNA polymerase II genes are the most potent obstacles for DNA replication in wild type yeast cells. Outside of the lab, Virginia Zakian is known for her many efforts to support and promote women and minorities in science.
Honors & Awards
- Featured speaker, EMBO conference Telomeres, Telomerase and Disease, Brussels, Belgium
- Keynote speaker, GRS for Gordon Res Conf., Chromosome Dynamics
- Keynote speaker, EMBO workshop, Telomere chromatin and telomere fragility,, Singapore
- Keynote speaker, Annual meeting, Swedish Society Biochemistry, Biophysics and Molecular Biology, Marstrand, Sweden
- Speaker Annual NIA IRP Postbac Day, National Institutes of Aging
- Diamonds are Forever: Celebrating First 75 Years, Princeton Adult School
- Barnum Museum Lecture, Tufts University
- President's Lecture Series, Princeton University
- Leading Edge Lecture, City of Hope
- Keynote speaker, Anat Krauskopf Symposium, Tel Aviv IS,
- Magni Lecture, Milan, Italy
- Barnum Museum Lecture, Tufts University
- President’s Lecture series, Princeton University
- Keynote Speaker, College of NJ, Advancement program Symposium
- Danny Kaye Lecture, St. Jude’s, Memphis
- Wall of Fame, Upper Darby Sr HS, Upper Darby PA
- Second Annual Athena Lecture, Royal Society, London
- Honors Program Lecture, NYU School of Medicine
- Distinguished Lecture, Lawrence Berkeley National Laboratory, Life Sciences Division
- Merit Award, NIGMS of the National Institutes of Health
- Elkin Distinguished Lectureship, Winship Cancer Center, Emory University
- Harold Varmus’ NIH Wednesday Afternoon Lectureship, National Institutes of Health
- Distinguished Lecture Series, NIEHS
- June Wood Lecture, Indiana University
- Blaffer Seminar, University of Texas M.D. Anderson Cancer Center
- Women in Cell Biology, Senior Woman Award, American Society of Cell Biology
- Fellow, American Academy of Microbiology
- Travel Fellowship, Ministry of Education, Japan
- Fellow, American Academy for the Advancement of Science
- B.S., Biology, Cornell University
- Ph.D., Biology, Yale University
- 1.Garcia D, Leach R, Wadsworth G, Choudhary K, Li H, Aviran S, Kim H, Zakian V. Stability and nuclear localization of yeast telomerase depend on protein components of RNase P/MRP. Nat Commun. 2020;11(1):2173. PMCID: PMC7195438
- 1.Chen C-F, Pohl T, Chan A, Slocum J, Zakian V. Saccharomyces cerevisiae Centromere RNA Is Negatively Regulated by Cbf1 and Its Unscheduled Synthesis Impacts CenH3 Binding. Genetics. 2019;. PMID: 31391265
- 1.Geronimo C, Singh S, Galletto R, Zakian V. The signature motif of the Saccharomyces cerevisiae Pif1 DNA helicase is essential in vivo for mitochondrial and nuclear functions and in vitro for ATPase activity. Nucleic Acids Res. 2018;46(16):8357–8370. PMCID: PMC6144861
- 1.Greider C, Hopkins N, Steitz J, Amon A, Asai D, Barres B, Bass B, Bassler B, Birgeneau R, Bjorkman P, Botchan M, Brugge J, Cech T, Colwell R, Craig N, deLange T, Eisen M, Gottesman S, Green R, Handelsman J, Kimble J, King M-C, Lehmann R, Marder E, Mullins D, O’Shea E, Schmid S, Seydoux G, Spradling A, Storz G, Szostak J, Telesnitsky A, Tilghman S, Tjian R, Vale R, Wolberger C, Zakian V. Not just Salk. Science. 2017;357(6356):1105–1106. PMID: 28912235
- 1.McDonald K, Guise A, Pourbozorgi-Langroudi P, Cristea I, Zakian V, Capra J, Sabouri N. Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity. PLoS Genet. 2016;12(9):e1006238. PMCID: PMC5017727
- 1.Lin K-W, McDonald K, Guise A, Chan A, Cristea I, Zakian V. Proteomics of yeast telomerase identified Cdc48-Npl4-Ufd1 and Ufd4 as regulators of Est1 and telomere length. Nat Commun. 2015;6:8290. PMCID: PMC4579843
- 1.Webb C, Zakian V. Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding. Proc Natl Acad Sci U S A. 2015;112(36):11312–7. PMCID: PMC4568702
- 1.Stundon J, Zakian V. Identification of Saccharomyces cerevisiae Genes Whose Deletion Causes Synthetic Effects in Cells with Reduced Levels of the Nuclear Pif1 DNA Helicase. G3 (Bethesda). 2015;5(12):2913–8. PMCID: PMC4683662