My research interests are in genome plasticity and protein-DNA machines. Our work includes the study of mechanisms of prokaryotic and eukaryotic transposons, exploring how host cells control and respond to transposition, i.e. DNA repair, and using transposition to probe genome structure and function.

Mobile DNA
Transposons, i.e. pieces of DNA that can move from place to place, are ubiquitous. Strikingly almost 50% of the human genome is made up of sequences derived from transposable elements! We are studying several different elements from prokaryotes and eucaryotes. Despite their diverse origins, our interests in all of these of elements are very similar: how do these reactions occur at the molecular level? What are the protein-DNA and protein-protein interactions that mediate transposition? How do host proteins influence transposition? Host proteins are both involved directly in transposition and also likely can control the frequency of transposition by, for example, influencing the level of active transposase. Transposition results in DNA damage, for example, the gap left in a donor site when an element excises. We also want to understand how the host responds to and repairs this break Our approaches to studying these different mobile elements are also similar: we have established in vitro transposition systems using purified proteins such that we can use a wide variety of biochemical, biophysical and structural methods to dissect these reactions. We also use genetics to study the element in its host and to isolate interesting host and transposition machine mutants.

hAT Family Transposons
One aspect of our work is focused on several members of the hAT family that is widespread in eukaryotes, being found in worms, flies, plants, fish and mammals including humans. Notably the hAT family includes Ac, the maize Ac element that McClintock discovered. Our in vitro analysis of the insect hAT element Hermes has lead to the first crystal structure of a eukaryotic transposase. This element is of particular interest because its mechanism of DNA breakage and joining is very closely related to how DNA breakage events occur during the V(D)J reactions that underlie the assembly of active immunoglobulin receptor genes. Our work also revealed that despite its differences in primary amino acid sequence from other elements, the hAT elements are part of the very widespread Retroviral Integrase Superfamily. We are isolating transposase mutants that transposase at higher than wild-type frequency for use in transgenesis experiments in diverse hosts including insects, fish and mammals. We are studying these elements in the yeasts S. cerevisiae and S. pombe, and in insect, fish and mammalian hosts.

Tn7
Another aspect of our work includes dissection of a sophisticated bacterial transposon called Tn7 that is of particular interest because of its target site selectivity. Tn7 recombination includes pathways in which the element is attracted to particular sites, for example, to a specific site in the bacterial chromosome, but also actively avoids inserting into other DNAs. The Tn7 transposition machinery is particularly elaborate: the reaction involves multiple proteins (4), the 2 transposon ends, the target DNA and the essential cofactors ATP and Mg2+. We are particularly interested in understanding how ATP binding and hydrolysis by one of these proteins controls transposition. This "transposition switch" is analogous, for example, to the GTP switch that controls the RAS oncoprotein. We are studying Tn7 in E. coli.

The Host & Transposition
The host may influence transposition at many levels from controlling the amount of transposase to mediating the DNA repair reactions required to restore DNA to its intact duplex state. For example, the donor site contains a large double-strand gap from which the element excised and there are single-strand gaps between the transposon end and the target DNAs. How does a cell respond to potentially lethal DNA breaks? Interestingly, the Ac family described above seems to use the Non-Homologous End Joining Pathway that also responds to ionizing irradiation. We are also interested in how the host controls the frequency of transposition. We are looking for host mutants that influence either control or repair to dissect these key steps.

Microbial Pathogenesis
Although E. coli can be a benign laboratory animal, there are also E. coli strains that are potent human pathogens. We are developing Tn7-based tools to discover bacterial pathogenic determinants.

Publications:

Stellwagen, A.E., Craig, N.L. (2001) Analysis of Gain of Function Mutants of an ATP-dependent Regulator of Tn7 Transposition. Journal of Molecular Biology 305(3):633-642.
PubMed Abstract

Kuduvalli, P.N., Rao, J.E., Craig, N.L. (2001) Target DNA Structure Plays a Critical Role in Tn7 Transposition EMBO Journal 20(4): 924-32.
PubMed Abstract

Peters, J.E., Craig, N.L. (2001) Tn7 recognizes target structures associated with DNA replication using the DNA binding protein TnsE. Genes & Development 15(6):737-747.
PubMed Abstract

Peters, J.E., Craig, N.L. (2001) Tn7: Smarter Than We Thought. Nat Rev Mol Cell Biology 2:806-814 Review.
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Craig, N.L., Craigie, R., Gellert, M., Lambowitz, A., Editors (2002) Mobile DNA II American Society for Microbiology, Washington DC

Skelding, Z., Sarnovsky, R., Craig, N.L. (2002) Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation EMBO J 21(13):3494-3504.
PubMed Abstract

Skelding, Z., Queen-Baker, J., Craig, N.L. (2003) Alternative Interactions between the Tn7 Transposase and the Tn7 Target DNA Binding Protein Regulate Target Immunity and Transposition EMBO J 22(21):5904-5917.
PubMed Abstract

Castano, I., R. Kaur, S. Pan, R. Cregg, A. De Las Penas, N. Guo, M.C. Biery, N.L. Craig, B.P. Cormack (2003) Tn7-based Genome-wide Random Insertional Mutagenesis of Candida glabrata Genome Res 13:905-915.
PubMed Abstract

Ronning, D.R., Y. Li, Z.N. Perez, P.D. Ross, A.B. Hickman, N.L. Craig, F. Dyda (2004) The carboxy-terminal portion of TnsC activates the Tn7 transposase through a specific interaction with TnsA EMBO J 23(14):2972-2981.
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Zhou, L., R. Mitra, P.W. Atkinson, A.B. Hickman, F. Dyda, N.L.Craig (2004) Transposition of hAT elements links transposable elements and V(D)J recombination Nature 432:995-1001.
PubMed Abstract

Kuduvalli, P.N., R. Mitra, N.L. Craig (2005) Site-specific Tn7 Transposition into the Human Genome Nucleic Acids Research 33:857-863
PubMed Abstract

Hickman, A.B., Perez, Z.N., Zhou, L., Musingarimi, P., Ghirlando, R., Hinshaw, J.E., Craig, N.L. and Dyda, F. (2005) Molecular architecture of a eukaryotic DNA transposase. Nat Struct Mol Biol 12(8):715-721.
PubMed Abstract