Postdoctoral, graduate and technical positions.
Studies of the molecular mechanisms of DNA replication.
Studies of the mechanism of origin activation in Herpes Simplex Virus.
Studies of the molecular mechanics of DNA repair.
Studies of telomere structure and function.
Studies of bio-molecules trapped in ancient salt deposits.
How to apply for a position in the Griffith laboratory.

Postdoctoral, graduate and technical positions.

There are always exciting research opportunities awaiting and positions available in our laboratory at technician, graduate student and postdoctoral levels. Below we have summarized the major projects ongoing in the laboratory. Information on applying is at the end of this section.

Advantages of joining our laboratory. Direct transmission electron microscopy has always been a powerful means of studying DNA and DNA protein complexes. In the past it provided the first demonstration of supercoiling of DNA, was used to discover RNA splicing, to reveal the nucleosome, and to show how DNA is bent by phased tracts of adenines. More recently it was used to discover that telomeres are arranged into loops. This technology is more and more in demand as molecular biologic studies across the world are moving toward understanding how complex DNA-protein machines are built and drive fundamental reactions in replication, transcription, and repair. Our laboratory has pioneered these methods and developed ways of combing this information with parallel biochemical studies. Students and fellows in our laboratory thus gain training which is unique both in the EM methods and in solid DNA-protein biochemistry. This has placed them in a particularly advantageous position in their career development.

Graduate Student positions. Frequently foreign students ask if it is possible to directly apply to an individual laboratory for admission. At the University of North Carolina, this is not possible. Students must be admitted through one of nearly a dozen different research departments. Once the student has been admitted, then he or she is able to select from many different laboratories in which to work. These departments include Genetics and Molecular Biology, Biochemistry, Microbiology and Immunology, Pathology, Cell Biology, and Pharmacology among others. Information about application can be found on the UNC Graduate Web Page: http://gradschool.unc.edu

Technician Positions. We frequently have openings for technicians in the laboratory. Strong preference is given to candidates with tissue culture or DNA cloning experience. In general at least 1 year of full time laboratory experience in the US or a masters degree is required. Technicians are treated as equal members of the laboratory and are expected to present their work in the weekly laboratory meetings and are frequently sent to national meetings. Their names are included on papers when they have made a substantial contribution to the work. Queries should be sent to the email address at the end of this section or by mail to Dr. Griffith at the Lineberger Comprehensive Cancer Center, Mason Farm Road, Chapel Hill, NC 27599.

Postdoctoral Positions. We are presently seeking fellows to join our laboratory in each of our major research topics described below. Salaries generally follow the NIH scale. The research environment at the Lineberger Comprehensive Cancer Center for fellows is superb with special seminar programs and retreats for fellows, as well as tutorials on grant writing and how to apply for faculty positions. Each fellows works on a specific project of his/her own and one which does not compete with projects of other laboratory members. Because we are always engaged in collaborations with other laboratories in the US and abroad, fellows are involved in one or more of these collaborations. This provides contacts with other labs, and provides additional publications to further build a resume.

The living environment. The Research Triangle area provides one of the most attractive areas to live in the US with costs of housing, utilities, food, and car insurance lower than it is in the large urban areas. Quality apartments close to UNC can be found for $650/month for a one bedroom apartment. Frequently fellows are able to live close enough to take a bus or bicycle to the lab. There are many recreational activities including water sports on the nearby Jordan Lake, horse riding, and numerous cultural activities at UNC, Duke and in Raleigh. More information can be found at (www.triangle.citysearch.com). Application for a position in our laboratory is described at the end of this section.

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Studies of the molecular mechanisms of DNA replication.

Analysis of single molecules by transmission electron microscopy (EM) provides a powerful means of studying the mechanics of DNA replication. Input from EM is as important to understanding large scale structural questions as obtaining Xray structures is to showing how polymerases catalyze DNA synthesis. While a detailed understanding of the mechanics of replication has been built up through a combination of biochemical and structural approaches, many key questions remain unanswered. Two in particular are how the architecture of the DNA strands at the fork facilitates the coupling of leading and lagging strand replication, and how origins are activated in the cell. The recent purification and characterization of the proteins which catalyze replication in a variety of prokaryotic and eukaryotic systems makes it possible to reconstruct the key steps in vitro using defined DNA templates and proteins. This allows us to study intermediates in these reactions at the single molecule level using EM. Our approach is to examine the structure of a statistically large number of replicating molecules and then to combine this information with data about the average population derived from biochemical assays. No single system will provide all of the structural answers, and it will be critical to compare results across a number of systems to determine which features are fundamental to all. We have established a highly interactive program with laboratories expert in specific replication systems: the yeast ORC (origin recognition complex) with Steve Bell (MIT), the human papillomaviruses with Tom Broker and Louise Chow (UAB), and T7 phage replication with Charles Richardson (Harvard). This has led to a focused, highly productive program in which we have obtained important structural understandings beyond that of the individual systems. The information flow among the four groups has greatly benefited the efforts of each. There has been a continual series of meetings with each group and beginning this year, all 4 groups will meet once a year. This is an open effort: new collaborations are welcomed as long as they are directly related to the goals of this grant and resources are available. In addition our own work on T4 and HSV-1 replication provides the core of this effort.

Specific areas being investigated:

I. Architecture of origin complex in yeast. Using several high resolution EM techniques, the structure of the yeast Origin Recognition complex (ORC) is being examined both alone and bound to yeast ARS DNA elements. A molecular pointer is being designed to localize individual subunits of the ORC, each individually tagged with biotin. EM will then used to explore the unwinding of the ARS by the ORC and other factors.

2. Origin opening and activation in HPV. The nature of eukaryotic origin recognition and opening is being probed using the human papillomavirus as a model. The ability of the viral E1 protein to unwind the origin in a reaction facilitated by E2 and host chaperone proteins is being examined using a combination of EM and biochemical tools. The role of host DNA polymerase alpha and cellular cyclin E and cdk2 kinase will be probed. Future-looking experiments will be initiated using cell extracts and SV40 and HPV origin based plasmids to obtain basic information about the folding of the DNA strands at a moving eukaryotic replication fork.

3. Architecture of the moving replication fork in the T7 and T4 systems. This long standing effort to understand the basic looping of the lagging strand at a moving replication fork will continue to be probed using the simple and well characterized T7 and T4 replication proteins. We will use EM to focus on the question of how Okazaki fragment size is controlled and the role of the structural 'spools' created by the binding of single stranded DNA on the lagging strand by the T7 SSB. Using the more complex T4 replication system we will continue our efforts to establish the generality of the looping of the lagging strand as a means of coupling leading and lagging strand replication. The nature of the loop and proteins required for its formation will be examined.

Selected References:

Kyusung Park, Zeger Debyser, Stanley Tabor, Charles C. Richardson, and Jack D. Griffith. Formation of a DNA Loop at the Replication Fork Generated by Bacteriophage T7 Replication Proteins. J. Biol. Chem. 273: 5260-5270. 1998.

Joonsee Lee, Paul Chastain II, Takahiro Kusakabe, Jack D. Griffith, and Charles C. Richardson. Coordinated Leading and lagging strand DNA synthesis on a mini-circular template. Molecular Cell. 1: 1001-1010. 1998.

Daniel G. Lee, Alexander M. Makhov, Richard D. Klemm, Jack D.Griffith, and Stephen P. Bell. Regulation of ORC conformation and ATPase Activity: differential effects of single-stranded and double-stranded DNA binding. EMBO J. 19: 1-9, 2000.

Biing Yuan Lin, Alexander M. Makhov, Jack D. Griffith, Thomas R. Broker and Louise T. Chow. Chaperone Proteins Abrogate the Inhibition of the Human Papillomavirus E1 Replicative Helicase by the HPV E2 Protein. Molecular Cell. Biol. 22: 6592-6604. 2002.

Paul D. Chastain I, Alexander M. Makhov, Nancy G. Nossal, and Jack D. Griffith. Analysis of the Okazaki Fragment Distributions along Single Long DNAs Replicated by the Bacteriophage T4 Proteins. Molecular Cell. 6: 803-814, 2000.

Paul D. Chastain, Alexander M. Makhov, Nancy G. Nossal, and Jack Griffith. Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage T4 proteins. J. Biol. Chem. 278:21276-21285, 2003.

Lisa F. Rezende, Smaranda Willcox, Jack D. Griffith, and Charles C. Richardson. A Single-stranded Binding protein of Bacteriophage T7 Defective in DNA annealing. J. Biol. Chem. 278: 29098-29105, 2003.

Paul D. Chastain II, Jayson L. Bowers, Daniel G. Lee, Stephen P. Bell, and Jack D. Griffith. Mapping Subunit Location on the S. cerevisiae ORC free and Bound to DNA using a Novel Nanoscale Biopointer. J Biol Chem. 279: 36,354-36, 362. 2004

Nancy G. Nossal, Alexander M. Makhov, Paul D. Chastain, II, Charles E. Jones, and Jack D. Griffith. Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers. J. Biol. Chem. 282: 1098-1108, 2007.

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Studies of the mechanism of origin activation in Herpes Simplex Virus.

Herpes Simplex type-1 virus (HSV-1) provides an excellent system for study of the early replication steps in a mammalian system. HSV-1 genome encodes seven proteins required for origin-dependent DNA replication consisting of a DNA polymerase and its accessory protein, a heterotrimeric helicase-primase, a single-strand (ss) DNA-binding protein (ICP8) and an origin-binding protein, UL9 protein. HSV-1 contains three functional origins of DNA replication. One, oriL, is present in the long unique segment of the genome while the other highly homologous origin, oriS, is present twice in the repeat region flanking the short unique segment. The minimal functional oriS sequence (79 bp) consists of a 45 bp palindrome containing a central AT-rich element flanked on each side by two high affinity UL9 protein-binding sites designated box I and box II. A third weaker UL9 protein-binding site, box III, is located adjacent to box I . The goal of this program is to understand the DNA-protein interactions that govern the activation of the two HSV-1 replication origins. Current studies are focusing on the origin binding protein, UL9, and the HSV-1 single strand binding protein ICP8. In addition, we are now in a position to assemble both origin containing DNAs into chromatin using the Drosophila chromatin assembly factors. This will be done with oriL DNA which contains specific motifs (the GRE) for hormone binding factors. Understanding how the origins are activated in the latent state will require understanding the role of chromatinization.

UL9 protein is a DNA dependent NTPase and helicase and contains six motifs characteristic of the superfamily of DNA helicases. With DNA containing oriS box I, specific binding of UL9 is observed which stimulates the UL9 ATPase activity at least 20-fold over the non-origin ds and ss DNAs. Electron microscope studies from our laboratory confirmed this, revealing a particle with the mass consistent with a double dimer assembled at oriS., In our EM studies, in the absence of ATP the DNA was observed to bend linear DNA about the UL9 double-dimer by ~90 degrees. Upon addition of ATP, DNA stem-loop structures encompassing many hundreds of nucleotides were seen extruded from the UL9 complex at oriS. The amount of DNA in the stem-loops increased with incubation time and mapping studies showed that the unwinding was bi-directional, centering about oriS. The DNA in the stem-loops frequently appeared as highly condensed rods and analysis showed that the DNA in them was partially single stranded and partially double stranded.

Specific project goals: We have now isolated all of the HSV-1 proteins which catalyze Herpes replication and these proteins are in hand in sufficient amounts to carry out studies similar to what we have been able to do in the T4 and T7 systems. These studies will for the first time show whether or not there is a trombone looping mechanism in eukaryotes. Further it will help define the role of each of these proteins in replication, information essential to the design of new anti-viral drugs.

Selected References:

Alexander Makhov, Paul Boehmer, I. Robert Lehman and Jack Griffith. The Herpes Simplex Virus type I UL9 protein carries out origin specific DNA unwinding and forms stem/loop structures. EMBO J. 15: 1742-1750, l996.

Alexander Makhov, Paul Boehmer, I. Robert Lehman and Jack Griffith. Visualization of the Unwinding of Long DNA Chains by the Herpes Simplex Virus type-I UL9 and ICP8. J. Mol. Biol. 258: 789- 799, 1996.

Nina B. Reuven, Smaranda Willcox, Jack D. Griffith, and Sandra K. Weller. Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nucleases. J Mol Biol. 342: 57-71. 2004

Alexander Makhov and Jack Griffith. Visualization of the Annealing of Complementary Single-stranded DNA Catalyzed by the Herpes Simplex Virus Type 1 ICP8 SSB/Recombinase. J Mol Biol. 355:911-22. 2006

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Studies of the molecular mechanics of DNA repair.

Loss or alteration of p53 function occurs in about half of all human cancers. The ongoing focus of our research continues to be understanding how p53 and the mismatch repair (MMR) proteins interact with DNA possessing simple lesions or large damage-containing structures and signal damage. Recently our studies revealed a surprising overlap in the binding of p53 and the MMR proteins to complex DNA structures as well as some simple lesions, in particular bulged bases and some base/base mismatches. Further, there may be synergistic interactions between p53 and MSH2-MSH6. By using a combination of biochemical tools and direct electron microscopic (EM) visualization, we are able to approach questions which cannot be addressed easily by other experimental means. One poorly understood question is how cells sense the presence of large damaged DNA structures, or potentially lethal DNA entanglements. Examples include large intertwined DNAs-- which if not resolved might lead to chromosome loss, Holliday junctions containing clusters of bulged bases on one arm, or replication forks with damage on a newly synthesized strand. These structures must be resolved or repaired prior to chromosome segregation, and thus cell cycle checkpoints must be in place to monitor their presence. It is likely that p53 participates in some of these checkpoints. Our long term goal in this project is to construct large DNA molecules with multiple structural features, for example replication forks with specific lesions near by, and then to ask how p53 and the MMR proteins together with other cellular proteins interact with these structures. This is an ongoing project and we will continue our studies on the binding of p53 to simple lesion-containing DNAs, and initiate studies on the remodeling of complex DNAs containing multiple structural features by p53, the MMR proteins and other cellular factors.

Stemming from the discovery of p53 consensus binding sites in DNA near p53 regulated genes, and the finding that p53 is a potent transactivator, it was assumed that all p53 dependent functions would be mediated via transcriptional mechanisms. Over the past several years, however, a growing body of evidence has accumulated suggesting a more direct role of p53 in some processes. It is known that p53 binds single-stranded (ss) DNA and double-stranded (ds) DNA ends, both typical products of DNA damage. We then showed that p53 forms stable complexes at sites of insertion/deletion mismatches (bulged or looped out bases) in DNA, a finding which was surprising at the time and suggested that p53 may participate directly in signaling the presence of DNA damage. Recently we used filter binding and gel shift assays to measure the binding of p53 to all possible single base/base mismatches in otherwise simple linear DNA (see Degtyareva et al below).

The post replication mismatch repair (MMR) proteins carry out the primary repair of base/base mismatches and looped out bases left behind after a replication fork due to polymerase errors or when a Holliday junction has passed through two recombining DNAs that are similar but not identical. At a replication fork, the mismatch repair proteins must distinguish the newly synthesized DNA strand from the old parental strand and then correct the mismatches to preserve the sequence fidelity of the parental DNA. In E. coli, this is accomplished via a methylation-based signaling system. The MutS protein recognizes the methylation signal, and along with the MutH and L proteins directs repair such that the fidelity of the old strand is maintained. As the signals may be spaced kilobases from a mismatch, a question was how this signaling could occur over such a long distance. In collaboration with the Modrich group we showed that the MutS protein binds directly to the mismatch in long DNAs, in the presence of ATP, a DNA loop is generated as the DNA strands are drawn in from both directions. The loops grow in size both with time and the hydrolysis of ATP.

In eukaryotes, the MutS homolog is represented by a set of at least 3 proteins, MSH2, MSH3 and MSH6. Heterodimers of MSH2 with 3 and 6 (MSH2-MSH3 or MSH2-MSH6) are found in vivo with the MSH2-MSH6 complex being the major species. These two heterodimers have overlapping but different specificity with the MSH2-MSH6 complex recognizing base/base mismatches and small loops and the MSH2-MSH3 complex recognizing base/ base mismatches and larger loops. The Mut L and H homologs are termed PMS1 and MLH1. Their role at a mechanistic level is less well understood.

Questions to be addressed in these studies

1. What is the effect of site specific modifications of p53 on its ability to recognize replicative and oxidative damage in DNA?

Filter binding and gel shift methods provide a means to quantify the binding of human p53 to linear DNAs containing base/base mismatches, extrahelical bulges and oxidative damage. p53 is being prepared in our laboratory routinely in SF9 insect cells. Using the purified protein, the effect of p53 phosphorylation at specific sites can be examined using kinases and phosphatases to either remove phosphate groups or to carry out site specific modifications. These studies have the potential of linking protein-specific modifications to the ability of p53 to respond to different lesions in DNA.

2. Is there long range communication by p53 and hMSH2-hMSH6 on Holliday junctions containing damage?

The way in which signaling occurs between lesions left behind a moving Holliday junction and the junction itself can be examined by generating Holliday junctions containing lesions at variable distances from the cross-over. We have recently developed means of generating these complex and very interesting templates. The hypothesis that p53 or hMSH2-hMSH6 can simultaneously bind both the damage site and the junction to generate a loop can now be examined for the first time and to test the hypothesis that p53 and the MMR proteins to communicate at a distance by an active remodeling of a complex DNA structure.

3. Does long range DNA looping occur linear DNAs and at replication forks containing damage at a distance mediated by p53 or MSH2/MSH6 action? We can now generate DNAs containing a replication fork and damage on the leading or lagging strands. These DNAs can be used to examine the mechanism by which damage is signaled between the fork and site(s) of damage by p53 and by the MMR proteins. In vitro replication systems are available through our replication studies to help determine if mutS,H&L will halt progress of the replication fork if there is damage in the preceding DNA strands.

Selected References:

Suman Lee, Brian Elenbaas, Arnold Levine and Jack Griffith. p53 and its 14 kDa C terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell 81: 1013-1020, 1995.

Eric Alani, Suman Lee, Michael Kane, Jack Griffith, and Richard Kolodner. Saccharomyces cerevisiae MSH2, a mispaired base recognition protein also recognizes Holliday junctions in DNA. J. Mol. Biol. 265: 289-301. 1997.

Gerald T. Marsischky, Suman Lee, Jack Griffith, and Richard D. Kolodner. Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage resolution enzymes. J. Biol. Chem. 274. 7200-7206. 1999.

Natalya Degtyareva, Deepa Subramanian, and Jack D. Griffith. Analysis of the binding of p53 to DNAs containing mismatched and bulged bases. J. Biol. Chem. 276: 8778-8784. 2001.

Rachel Stansel, Deepa Subramanian, and Jack D. Griffith. p53 Binds Telomeric Single Strand Overhangs and t-loop Junctions in Vitro. J. Biol. Chem. 277, 11,625-11, 628, 2002.

Keziban Unsal-Kacmaz, Alexander Makhov, Jack D. Griffith, and Aziz Sancar. Preferential binding of ATR protein to UV-damaged DNA. Proc. Natl. Acad. Sci. USA. 99: 6673-6678, 2002.

Funda Sar, Laura A Lindsey-Boltz, Deepa Subramanian, Deborah L. Croteau, Stephanie Q. Hutsell, Jack D. Griffith, and Aziz Sancar. Human Claspin is a Ring- shaped DNA binding Protein with High Affinity to Branched DNA Structures. J Biol. Chem. 279: 39,289-39,295. 2004

Deepa Subramanian and Jack D. Griffith. Modulation of p53 binding to Holliday junctions and 3-cytosine bulges by phosphorylation events. Biochemistry. 44: 2536-2544, 2005.

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Studies of telomere structure and function

Telomeres are believed to represent a molecular clock that regulates the number of cell divisions allowed before cellular senescence. This regulation appears to depend on the length of the telomeric DNA and thus by inference, must also depend on the physical structure of the telomere as it is folded into a loop and then further compacted by telomere specific proteins such as TRF1 and TRF2. Understanding the structure of this particle, how it changes with the age of the cell, and how it depends on the function of specific aging-related genes is a central goal of this study.

Over the past several years we collaborated with Dr. Titia de Lange at the Rockefeller University to understand how two proteins which bind mammalian telomeric DNA, TRF1 and TRF2, organize telomere architecture. From our EM examination of TRF2-DNA complexes and consideration of how recombination proteins such as recA would interact with DNA having the sequence and architecture of a mammalian telomere, a novel proposal was made. We suggested that the protruding 3' single-stranded overhang of the telomere would fold back and invade the preceding telomeric duplex tract to form a classic D-loop. Incubations of a model telomere DNA with purified TRF2 protein revealed looped forms in vitro. This was followed by the isolation of psoralen crosslinked telomeric DNA and EM visualization which revealed mammalian telomeres arranged into giant duplex loops (termed t-loops). Telomere looping may be a general paradigm since it has now been observed in several lower eukaryotes. This opened a new vista into telomere structure and posed critical questions whose answers will ultimately describe the processes controlling the aging clock as well as progression of cells to an oncogenic state. We will continue this productive, enjoyable collaboration, but having established our own directions, roughly half of of our work will be devoted to studies from this laboratory alone, or with other investigators.

Presently EM provides the only definitive assay for the arrangement of telomeres into loops and there are many pressing experiments to be done relating cellular aging to the properties of t-loops. One research goal is to develop non-EM based assays for t-loops. EM however will remain an essential primary tool for some time. We will grow human fibroblasts in culture through multiple generations beginning with isolation from newborn foreskin and ending in senescence. The size distribution and frequency of t-loops will be monitored throughout this process, with particular emphasis on the later stages as the cell approach senescence. EM analysis of telomere looping can be done from a single mouse liver, opening the door to studies that take advantage of the many transgenic mouse lines which contain mutations in genes related to telomere maintenance and aging. T-loop preparations will be made from mice of successive breeding generations that lack functional telomerase (in collaboration with Dr. Carol Greider) and the size of the loops determined at each generation. Werner's syndrome is a premature aging syndrome and the affected gene codes for a DNA helicase that could interact with the t-loop junction. We will determine whether cells from these mice have normal sized telomere loops and in work with Dr. Curt Harris will determine how purified Werner's helicase interacts with model t-loops formed in vitro. In studies with Dr. Titia de Lange, we will determine whether t-loops persist after the functional removal of TRF2 from cells. Their studies based on expression of dominant negative forms of TRF2 suggest that looping may be disrupted in the absence of TRF2 action. In other studies with her laboratory, human cells will be synchronized, and at various points through the cell cycle we will examine the cells for the presence and size distribution of telomere loops.

The discovery of t-loops led us to propose that the mammalian telomere is organized into a highly ordered, condensed nucleoprotein particle. In this model, the normal chromosomal proteins (histones) would induce the first level of DNA packing into chromatin particles termed nucleosomes, followed by further condensation by TRF1 and TRF2. This compact particle could sequester the t-loop junction from nucleases such as the MRE11/Rad50/NBS1 complex (presumably the loop would open at S phase for replication). When cells age and the telomeric DNA becomes critically short, the telomere particle would, we suggest, be too small to provide adequate protection the t-loop junction. We estimate that this would occur when the telomere shortens to ~2-3 kb (15 or fewer nucleosomes). This is, indeed, close to the critical length of human telomeres at senescence. The histones bind telomeric DNA, but not as strongly as normal sequence DNA and human telomeres exhibit a nuclease digestion pattern suggestive of a tighter than usual nucleosome spacing. The uniform sequence of telomeric DNA does not present energy barriers to the histone particles in their sliding along DNA and this could generate an extremely regular packaging of the telomeric DNA which would be further condensed by TRF1 and TRF2. Our goal is to reconstitute telomeric DNA into a chromosomal structure by incubating high molecular weight human telomeric DNA with purified histone proteins, as well as TRF1 and TRF2. This will be done using a new chromatin assembly system derived from Drosophila cells. Several modes of EM analysis will be employed including cryo EM. These studies will be complemented by nuclease and chemical probing. We believe this to be a compelling model which could explain how the cell measures telomere length and thus regulates cellular life span. Reconstitution experiments such as these are the most direct way to test this model and EM provides the only clear means of examining such large structures.

Selected References:

Jack Griffith, Laurey Comeau, Soraya Rosenfield, Rachel Stansel, Heidi Moss, Alessandro Bianchi, and Titia de Lange. Mammalian telomeres end in a large duplex loop. Cell: 97, 503-514. 1999.

Alessandro Bianchi, Rachel Stansel, Lousie Fairall, Jack D. Griffith, Daniela Rhodes, and Titia de Lange. TRF1 binds a bipartite site with extreme spatial flexibility. EMBO J. 18: 5735-5744. 1999.

Lubomir Tomaska, Alexander M. Makhov, Jozef Nosek, Blanka Kucejova, and Jack D. Griffith. Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis. J Mol. Biol., 305: 61-69 2001.

Lubomir Tomaska, Jozef Nosek, Alexander M. Makhov, Andrea Pastorakova, and Jack D. Griffith. Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: Potential involvement in telomere maintenance. in press, Nucleic Acids Research, 28: 4479-4487, 2000.

Jorge L Munoz-Jordan, George A.M. Cross, Titia de Lange, andJack D. Griffith. T-loops at trypanosome telomeres. In press, EMBO J. 2001 Anthony J. Cesare, Nancy Quinney, Smaranda Willcox, Deepa Subramanian and Jack D. Griffith. Telomere Looping in P. savitum (common garden pea). The Plant Journal. 36: 271-279. 2003.

Lubomir Tomaska, Smaranda Willcox, Judita Slezakova, Jozef Nosek, and Jack D. Griffith. Taz1 binding to a fission yeast model telomere: formation of t-loops and higher order structures. J Biol Chem, 279: 50764-50772, 2004.

Anthony J. Cesare and Jack D. Griffith. Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops. Mol. Cell. Biol 24: 9948-9957. 2004.

Jozef Nosek, Adriana Rycovska, Judita Slezakova, Alexander M. Makhov, Jack D. Griffith, and Lubomir Tomaska. Amplification of telomeric arrays via rolling-circle mechanism. J. Biol. Chem. 10840-10845. 2005.

Cindy Groff-Vindman, Anthony J. Cesare, Shobhana Natarajan, Jack D. Griffith and Michael J. McEachern Recombination at long mutant telomeres produces tiny ss and ds telomeric circles. Mol. Cell. Biol. 11: 4406-4412, 2005

Electron Microscopic Visualization of Telomerase from Euplotes aediculatus Bound to a Model Telomere DNA Nicole Fouché, Ian K. Moon, Brian R. Keppler, Jack D. Griffith and Michael B. Jarstfer. Biochemistry 45: 9624-9631. 2006

Nicole Fouche, Sezgin Ozgur, Desmandia Roy and Jack D. Griffith. Replication fork regression in repetitive DNAs. Nucl. Acids. Res. 34: 6044-6050, 2006

Nicole Fouche, Anthony J. Cesare, Smaranda Willcox, Sezgin Ozgur, Sarah A. Compton and Jack D. Griffith, The Basic Domain of TRF2 Directs Binding to DNA Junctions Irrespective of the Presence of TTAGGG Repeats. J. Biol. Chem. 281: 37486-37495, 2006

Sarah A Compton, Jun-Hyuk Choi, Anthony Cesare, Sezgin Ozgur, Jack Griffith. Xrcc3 and Nbs1 are required for Telomere Length Maintenance and Production of Telomeric Circles in Human ALT cells. Cancer Res, Feb 15 2007; 67(4)

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Studies of bio-molecules trapped in ancient salt deposits.

This is a new exploratory project and funding from the NSF is being sought. Therefore, it will be carried out as side projects by lab members until stable funding is in hand. None the less this work has great potential for opening a critical new door into understanding the nature of the biosphere 200+ million years ago. Over the past 2 years we have visited a deep mine in New Mexico (near Roswell) where transuranium waste is stored by the DOE. The mine is in a layer of halite (NaCl) crystals which were laid down in the early Permian period. We have brought back to UNC several hundred pounds of the halite and developed means of eliminating any modern biological material which might have contaminated the surface while being able to purify biological material and macromolecules that had been trapped in the halite. The result of this work has been the finding of a small amount of DNA which is likely highly crosslinked, and a large amount of cellulose microfibers. The cellulose microfibers are native in their structure and appearance. These molecules represent the oldest native biological macromolecules ever isolated on Earth. The possibility that cellulose may provide the ideal "paper trail" for detecting life on other planets has not escaped our thinking and we are persuing this avenue. A paper describing the ancient cellulose is under review.

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How to apply for a position in the Griffith laboratory.

To apply for a position, you can either write Dr. Griffith at the Lineberger Comprehensive Cancer Center, Mason Farm Road, University of North Carolina, Chapel Hill, NC 27599-7295, or send your application to us at the following email address: jdglab@email.unc.edu

For postdoctoral fellows, please include a description of your Ph.D thesis work, which study in our laboratory you are most interested in, and the name and contact information of 3 faculty members who can be asked for a reference. The University of North Carolina is an equal opportunity employer.

Language requirements. Because we use very complex and expensive electron microscopes and preparative equipment, members of the laboratory must be able to communicate well in English. For this reason, for individuals from mainland China, Dr. Griffith requires that they have spent at least 1 year in the US or in an English speaking laboratory before joining our research group.

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