Abstract
Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively. Here, we establish that both PCNA1 and PCNA2 form homotrimers that stimulate DNA synthesis by archaeal DNA polymerases B and D and ATP hydrolysis by the replication factor C complex. In exponentially growing cells, PCNA1 is abundant and present at an ~100-fold higher concentration than PCNA2 monomers. Deletion of TK0582 (PCNA2) had no detectable effects on viability or growth whereas repeated attempts to construct a T. kodakarensis strain with TK0535 (PCNA1) deleted were unsuccessful. The implications of these observations for PCNA1 function and the origin of the two PCNA-encoding genes in T. kodakarensis are discussed.
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Abbreviations
- PolB:
-
DNA polymerase B
- PolD:
-
DNA polymerase D
- PCNA:
-
Proliferating cell nuclear antigen
- RFC:
-
Replication factor C
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Acknowledgments
This work was supported by grant MCB-0815646 from National Science Foundation to ZK, and grants GM073336 to TJS, GM53185 to JNR and TJS and GH034559 to JH from the National Institutes of Health. Certain commercial materials, instruments, and equipment are identified in this paper to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the materials, instruments, or equipment identified are necessarily the best available for the purpose.
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Communicated by H. Atomi.
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Pan, M., Santangelo, T.J., Čuboňová, Ľ. et al. Thermococcus kodakarensis has two functional PCNA homologs but only one is required for viability. Extremophiles 17, 453–461 (2013). https://doi.org/10.1007/s00792-013-0526-8
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DOI: https://doi.org/10.1007/s00792-013-0526-8