Abstract
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 single-stranded DNA in the 5' to 3' direction and requires a 5' single-stranded overhang, or ss DNA 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.
