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 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.
Subject(s)
DNA Helicases , DNA Polymerase III , Escherichia coli Proteins , Escherichia coli , DNA Helicases/metabolism , DNA Polymerase III/genetics , DNA Replication , DNA, Single-Stranded , Escherichia coli/metabolism , Iron , Escherichia coli Proteins/metabolism , DNA RepairABSTRACT
Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme-DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein-protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein-protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading.
Subject(s)
Escherichia coli Proteins , DNA Replication/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , DNA Repair/geneticsABSTRACT
One-half of all new infections with the human immunodeficiency virus (HIV) and cases of acquired immunodeficiency syndrome (AIDS) occur in young people under age 25. While many markers for the spread of HIV/AIDS have been declining, the Centers for Disease Control and Prevention (CDC) reports no similar reduction in newly diagnosed HIV cases among youth. These findings point to the need for greater prevention and education efforts to help curb the spread of HIV/AIDS among the nation's youth. Community HealthCorps members working in community health centers provided HIV/AIDS prevention education to youth as part of their community service work. After HealthCorps members received training in the HIV/AIDS prevention curriculum, Be Proud! Be Responsible!, they recruited young people from their communities to participate in the program. HealthCorps members provided prevention education to students to ensure they acquired basic knowledge about HIV/AIDS on which to base personal decisions and actions. Staff of the Adolescent School Health Initiative of the National Association of Community Health Centers assessed the impact of the education on youth knowledge of HIV/AIDS, understanding of personal risk, their ability to refuse or negotiate safer sex, and proper use of condoms. This article highlights findings and summarizes lessons learned from the prevention education initiative.
