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Journal of Bacteriology, September 2008, p. 5766-5780, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.01930-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Engineering of Functional Replication Protein A Homologs Based on Insights into the Evolution of Oligonucleotide/ Oligosaccharide-Binding Folds ^,

Yuyen Lin,¹ Li-Jung Lin,¹ Palita Sriratana,² Kelli Coleman,¹ Taekjip Ha,^3,4,5 Maria Spies,⁶ and Isaac K. O. Cann^1,2,4*

Department of Animal Sciences,¹ Department of Microbiology,² Department of Physics,³ Institute for Genomic Biology,⁴ Howard Hughes Medical Institute,⁵ Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801⁶

Received 12 December 2007/ Accepted 18 June 2008

The bacterial single-stranded DNA-binding protein (SSB) and the archaeal/eukaryotic functional homolog, replication protein A (RPA), are essential for most aspects of DNA metabolism. Structural analyses of the architecture of SSB and RPA suggest that they are composed of different combinations of a module called the oligonucleotide/oligosaccharide-binding (OB) fold. Members of the domains Bacteria and Eukarya, in general, contain one type of SSB or RPA. In contrast, organisms in the archaeal domain have different RPAs made up of different organizations of OB folds. Interestingly, the euryarchaeon Methanosarcina acetivorans harbors multiple functional RPAs named MacRPA1 (for M. acetivorans RPA 1), MacRPA2, and MacRPA3. Comparison of MacRPA1 with related proteins in the publicly available databases suggested that intramolecular homologous recombination might play an important role in generating some of the diversity of OB folds in archaeal cells. On the basis of this information, from a four-OB-fold-containing RPA, we engineered chimeric modules to create three-OB-fold-containing RPAs to mimic a novel form of RPA found in Methanococcoides burtonii and Methanosaeta thermophila. We further created two RPAs that mimicked the RPAs in Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus through fusions of modules from MacRPA1 and M. thermautotrophicus RPA. Functional studies of these engineered proteins suggested that fusion and shuffling of OB folds can lead to well-folded polypeptides with most of the known properties of SSB and RPAs. On the basis of these results, different models that attempt to explain how intramolecular and intermolecular homologous recombination can generate novel forms of SSB or RPAs are proposed.

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* Corresponding author. Mailing address: Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Drive, Urbana, IL 61801. Phone: (217) 333-2090. Fax: (217) 333-8286. E-mail: icann{at}uiuc.edu

Published ahead of print on 27 June 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, September 2008, p. 5766-5780, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.01930-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.