Honors and Awards
Overview of the lab
Our laboratory is unique around the world in being able to combine sophisticated biochemistry, genetics, and molecular biology with the ability to directly visualize DNA, proteins, and DNA or RNA-protein complexes, as well as whole cells using high resolution electron microscopes. Most of the methods for preparing the samples are ones we developed and thus we have an expertise which few other groups possess. This has resulted in many research papers of great significance, including the first visualization of nucleosomes, the first visualization of bent DNA, and the discovery of telomere looping. We have a wealth of collaborations with laboratories world-wide which greatly enriches our research program and the training environment for laboratory members who frequently collaborate and publish with top groups elsewhere. I keep the lab relatively small (6 to 8 in addition to myself) so that I too can carry out experiments at the bench, which I have continued to do my whole career. All members of the laboratory, graduate students, fellows, and technicians are considered equals, and this year (2011) the whole group traveled to Alaska in the summer for a workshop on aging and DNA repair that we organized.
Electron microscopes: Our laboratory also serves as an EM core facility in the Lineberger Comprehensive Cancer Center at UNC. We have two 120 KV instruments, an FEI Tecnai12 and a Philips CM12. Both are fitted with state of the art Gatan real time and slow scan cameras. The CM12 has new cyrostages for cryoEM. In 2010 we were awarded an NIH grant for $300,000 to upgrade our cryoEM equipment and this allowed us to obtain an FEI Vitrobot, a robotic freezing instrument for cryoEM. We have three high vacuum systems including one for freeze-drying molecules and cells.
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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 biology 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 graduate programs. Once the student has been admitted, then he or she is able to select from many different laboratories in the departments of 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, 450 West Drive, 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 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 fellow 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 groups, and provides additional publication opportunities 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, bicycling, 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.
Our Research studies:
Specific areas being investigated:
1. 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. Opening of the origin by the action of the mcm2-7 helicase is being examined by EM.
2. Origin opening and activation in HPV. The nature of eukaryotic origin recognition and opening is being probed using the human papilloma virus 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. ()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 systems. We 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 synthesis. The nature of the loop and proteins required for its formation will be examined. Recent work has combined EM with the single molecule methods pioneered by Antoine van Oijen making this dual approach particularly powerful.
Selected References: (see also recent papers, 2009-2011 under publications).
Selected References: (see also
papers, 2009-2011 under publications).
HSV-1. 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. 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.
When cells are infected by HSV-1, the viral DNA is replicated in
condensed bodies in the cell nuclei (replication bodies or ND10
bodies). These bodies contain not only all of the replication factors,
but also many of the host, human DNA repair proteins. We will
purify these bodies from HSV-1 infected human cells (Vero), on sucrose
gradients. Using EM it will be possible to investigate their 3D
structure and composition (using gold tagged antibodies). This will
provide a critically needed view into the architecture and mechanism of
DNA replication factories in a human cell.
We have spend considerable effort over the years to solve the
structure of the filaments formed by the HSV-1 recombinase/SSB protein
called ICP8. The study with the Egelman laboratory (see recent papers)
provides the most detailed structure yet, and work to be published
shortly will refine these structures. In the classic thinking,
“proteins come to the DNA, act and leave”. More and more we
suspect that in the dense replication bodies in HSV-1 infected cells,
pre-formed filaments of ICP8 protein exist as protein scaffolds,
waiting for “DNA to come to the protein and then leave”. In this model
the DNA would meld into these protein scaffolds, undergo annealing and
recombination events and then be released. This new view will be tested
by a series of both in vitro and in vivo studies. If true, it would
greatly reshape our thinking of replication.
KSHV. Kaposi’s sarcoma
herpes virus (HHV8) is a newly discovered herpes virus linked to
several human cancers. There is a very active effort among several
groups at the Lineberger Comprehensive Cancer Center to investigate the
replication and life cycle of this virus. Our group has recently
isolated the KSHV SSB/recombinase (similar to ICP8) and has shown that
it can form protein filaments in solution (Ozgur, Damania, and
Griffith, 2011). We are currently further characterizing this protein
by biochemical approaches and will be carrying out mutant studies as
well as studies of how the origin binding protein of KSHV binds and
remodels the KSHV origin. Future work will entail the cloning and
characterization of each of the KSHV replication proteins.
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
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).
There are a large number of DNA repair factors that are now known to
interact alone and in concert with other repair and telomere-related
proteins on damaged DNA. We have isolated many of these factors and
have others available via collaborations. These include Rad51, the
Rad51 paralogs, the WRN and BLM helicases, polymerase beta, and BRCA2.
Understanding how these proteins alone and in combination interact at
sites of DNA damage is critical to our understanding of genomic repair
and genomic toxicology. In this regard, EM is unique in being able to
answer biochemical and structural questions that would be nearly
impossible to answer by any other approach. Our laboratory is also able
to engineer and build large complex DNA templates, kilobases in size
which have replication forks, Holliday junctions, and other structures.
These are unique templates for the EM studies and are much more
relevant to events in the cell than tiny DNAs generated from
Over the years we have 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, this P.I. made a novel proposal. I 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 mice, chickens, man, plants and 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.
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 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 cells 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 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 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 inducible knockout mice for TRF2 provide the ideal experimental system for deducing the true role of TRF2 in the cell.
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 at 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 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.
We have recently purified ample amounts of each of the telomere-associated proteins (shelterins) including hRAP1, Pot1, Tin2, TPP1 and are poised to generate combinations of these proteins bound to model telomere templates. Using a combination of EM and biochemical methods it will be possible to answer many basic questions about the role of these proteins in protecting the telomere.
A longstanding collaboration with Dr. Lubomir Tomaska at Comenius University in Brtatislava has provided the ideal venue for us to utilize the power of yeast genetics to answer basic questions of telomere function. These active studies are continuing.
To apply for a position, please send an initial inquiry and c.v. by email to Dr. Griffith email@example.com
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.