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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Primer Handoff between DNA Primase and DNA Polymerase α

DNA replication is a fundamental process. In eukaryotes, the first step in daughter strand synthesis is the generation of a short (~10 nt) RNA primer by DNA primase, followed by transfer of the primer to DNA polymerase α (Pol α) for extension by ~20 nts (1-5). Although the catalytic activity and structures of primase and Pol α have been extensively studied (2-5) the molecular mechanism of primer handoff between primase and Pol α remains largely unknown. We have proposed that redox switching of the [4Fe4S] cluster in primase is important for primer truncation and handoff to Pol α (1,4) Eukaryotic Pol α is composed of a catalytic subunit (p180) and a regulatory subunit (p68). The p180 subunit contains a [4Fe4S] cluster. Thus, we hypothesize that the redox partner of primase is Pol α and that the two [4Fe4S] clusters orchestrate the primer handoff between the two [4Fe4S] proteins using DNA-mediated charge transport chemistry. Here we present results using anaerobically purified human [4Fe4S] (C-terminal domain of p180 (p180C) in complex with p68. To the best of our knowledge, this is the first time that p180C-p68 is isolated with an [4Fe4S] cluster bound. The cluster loading can be improved to nearly 100% after initial purification using a reconstitution protocol. The protein exhibits electrochemical properties similar to the p58C [4Fe4S] domain of primase. Using multiplexed DNA-modified Au electrodes, we observe a large cathodic peak between –170 and –180 mV versus NHE by cyclic voltammetry only during the initial scan to negative potentials after oxidation by bulk electrolysis. As with p58C, the data are consistent with a model where p180C-p68 is more tightly associated with DNA in the oxidized state compared to the reduced state. We also present results from primer elongation assays, where truncated products are formed to a greater extent in the presence of the primase-p180C-p68 complex versus primase alone. When electrochemically oxidized p180C-p68 was assayed together with primase, the length of the products is similar to the products produced with primase alone. Together, these data are consistent with our hypothesis that the [4Fe4S] clusters in p180C-p68 and primase regulate the primer handoff from primase to Pol α.

References

    1 O'Brien, E.; Holt, M. E.; Thompson, M. K.; Salay, L. E.; Ehlinger, A. C.; Chazin, W. J.; Barton J. K. (2017). The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport. Science. 355: eaag1789.

    2 Burgers, P. M. J.; Kunkel, T. A. (2017). Eukaryotic DNA replication fork. Annu. Rev. Biochem. 86: 417-438.

    3 Pellegrini, L. (2012). The Pol α-primase complex. Subcell Biochem. 62: 157-69.

    4 O'Brien, E.; Salay, L. E.; Epum, E. A.; Friedman, K. L.; Chazin, W. J.; Barton, J. K. (2018). Yeast require redox switching in DNA primase. Proc. Natl. Acad. Sci. U. S. A. 115: 13186-13191.

    5 O'Brien, E.; Holt, M. E.; Salay, L. E.; Chazin, W. J.; Barton, J. K. (2018). Substrate binding regulates redox signaling in human DNA primase. J. Am. Chem. Soc. 140: 17153–17162.

Aoshu Zhong a
Lauren E. Salay b
Walter J. Chazin b
Jacqueline K. Barton a

aoshu_zhong-photo.gif

Aoshu Zhong is a post-doctoral fellow with Professor Jackie Barton at Caltech, and he will provide a short oral from the platform.

a Division of Chemistry and Chemical Engineering
California Institute of Technology
Pasadena, California 91125, US.

bDepartments of Biochemistry and Chemistry
Center for Structural Biology
Vanderbilt University
Nashville, TN 37240, US.

Email: azhong@caltech.edu