Single-stranded DNA binding protein hitches a ride with the Escherichia coli YoaA-χ helicase.

The Escherichia coli XPD/Rad3-like helicase, YoaA, and DNA polymerase III subunit, χ, are involved in E. coli DNA damage tolerance and repair. YoaA and χ promote tolerance to the DNA chain-terminator, 3 -azidothymidine (AZT), and together form the functional helicase complex, YoaA-χ. How YoaA-χ contributes to DNA damage tolerance is not well understood. E. coli single-stranded DNA binding protein (SSB) accumulates at stalled replication forks, and the SSB-χ interaction is required to promote AZT tolerance via an unknown mechanism. YoaA-χ and SSB interactions were investigated in vitro to better understand this DNA damage tolerance mechanism, and we discovered YoaA-χ and SSB have a functional interaction. SSB confers a substrate-specific effect on the helicase activity of YoaA-χ, barely affecting YoaA-χ on an overhang DNA substrate but inhibiting YoaA-χ on forked DNA. A paralog helicase, DinG, unwinds SSB-bound DNA in a similar manner to YoaA-χ on the substrates tested. Through use of ensemble experiments, we believe SSB binds behind YoaA-χ relative to the DNA ds/ss junction and show via single-molecule assays that SSB translocates along ssDNA with YoaA-χ. This is, to our knowledge, the first demonstration of a mechanoenzyme pulling SSB along ssDNA.

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

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.