Characterization of the Escherichia coli XPD/Rad3 iron-sulfur helicase YoaA in complex with the DNA polymerase III clamp loader subunit chi (χ).

Autor:	Weeks-Pollenz SJ; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA., Ali Y; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA., Morris LA; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA., Sutera VA; Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, USA., Dudenhausen EE; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA., Hibnick M; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA., Lovett ST; Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, USA., Bloom LB; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA. Electronic address: lbloom@ufl.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2023 Jan; Vol. 299 (1), pp. 102786. Date of Electronic Publication: 2022 Dec 09.
DOI:	10.1016/j.jbc.2022.102786
Abstrakt:	Escherichia coli YoaA aids in the resolution of DNA damage that halts DNA synthesis in vivo in conjunction with χ, an accessory subunit of DNA polymerase III. YoaA and χ form a discrete complex separate from the DNA polymerase III holoenzyme, but little is known about how YoaA and χ work together to help the replication fork overcome damage. Although YoaA is predicted to be an iron-sulfur helicase in the XPD/Rad3 helicase family based on sequence analysis, the biochemical activities of YoaA have not been described. Here, we characterize YoaA and show that purified YoaA contains iron. YoaA and χ form a complex that is stable through three chromatographic steps, including gel filtration chromatography. When overexpressed in the absence of χ, YoaA is mostly insoluble. In addition, we show the YoaA-χ complex has DNA-dependent ATPase activity. Our measurement of the YoaA-χ helicase activity illustrates for the first time YoaA-χ translocates on ssDNA in the 5' to 3' direction and requires a 5' single-stranded overhang, or ssDNA gap, for DNA/DNA unwinding. Furthermore, YoaA-χ preferentially unwinds forked duplex DNA that contains both 3' and 5' single-stranded overhangs versus duplex DNA with only a 5' overhang. Finally, we demonstrate YoaA-χ can unwind damaged DNA that contains an abasic site or damage on 3' ends that stall replication extension. These results are the first biochemical evidence demonstrating YoaA is a bona fide iron-sulfur helicase, and we further propose the physiologically relevant form of the helicase is YoaA-χ. Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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