The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein

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Abstract

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

Publications

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CDK1 Inhibition Targets the p53-NOXA-MCL1 Axis, Selectively Kills Embryonic Stem Cells, and Prevents Teratoma Formation

Stem Cell Reports 2015

Noelle E Huskey, Tingxia Guo, Kimberley J Evason, Olga Momcilovic, David Pardo, Katelyn J Creasman, Robert L Judson, Robert Blelloch, Sco...

Two miRNA Clusters Reveal Alternative Paths in Late-Stage Reprogramming

Cell Stem Cell 2014

Ronald J Parchem, Julia Ye, Robert L Judson, Marie F Larussa,Raga Krishnakumar, Amy Blelloch, Michael C Oldham, Robert Blelloch

MicroRNA-based discovery of barriers to dedifferentiation of fibroblasts to pluripotent stem cells

Nature Structural & Molecular Biology 2013

Robert L Judson, Tobias S Greve, Ronald J Parchem, Robert Blelloch

microRNA Control of Mouse and Human Pluripotent Stem Cell Behavior

Annual Review of Cell and Developmental Biology 2013

Tobias S Greve, Robert L Judson, Robert Blelloch

Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells

Nature Biotechnology 2011

Deepa Subramanyam, Samy Lamouille, Robert L Judson, Jason Y Liu, Nathan Bucay, Rik Derynck, Robert Blelloch

miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN-amplified neuroblastoma

Nature Medicine 2010

Alexander Swarbrick, Susan L Woods, Alexander Shaw, Asha Balakrishnan, Yuwei Phua, Akira Nguyen, Yvan Chanthery, Lionel Lim, Lesley J Ash...

Opposing microRNA families regulate self-renewal in mouse embryonic stem cells

Nature 2010

Collin Melton, Robert L Judson, Robert Blelloch

Embryonic stem cell-specific microRNAs promote induced pluripotency

Nature Biotechnology 2009

Robert L Judson, Joshua E Babiarz, Monica Venere, Robert Blelloch

The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein

Journal of Biological Chemistry 2008

Hirotaka Ebina, Atreyi Ghatak Chatterjee, Robert L Judson, Henry L Levin

The self primer of the long terminal repeat retrotransposon Tf1 is not removed during reverse transcription.

Journal of Virology 2006

Angela Atwood-Moore, Kenneth Yan, Robert L Judson, Henry L Levin

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