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<p>The <i>Escherichia coli</i> helicase, YoaA, and DNA polymerase III subunit, χ, form a complex (YoaA-χ) that promotes tolerance to the DNA chain-terminator 3ˈ-azidothymidine (AZT). Single-stranded DNA binding protein (SSB), which accumulates at stalled replication forks, also contributes to AZT tolerance through interactions with χ. Here we show that in vitro, χ mediates interactions between YoaA and SSB that modulate helicase activity in a substrate-specific manner with little effect on overhang DNA but inhibiting unwinding of forked DNA. SSB similarly affects the activity of the YoaA paralog, DinG. Single-molecule experiments show that SSB translocates with YoaA-χ, increasing both the lifetime and frequency of SSB binding events. Mutational analyses show that χ binds at the back of YoaA relative to the direction of translocation supporting a model in which YoaA-χ pulls SSB along DNA as it translocates. To our knowledge, this is the first demonstration of a mechanoenzyme pulling SSB along ssDNA.</p>

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The YoaA-χ helicase modulates the dynamics of single-stranded DNA binding protein on DNA

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月29日

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  • Savannah J. Weeks-Pollenz,
  • Matthew J. Petrides,
  • Kaylie A. Padgett-Pagliai,
  • Robert Davis,
  • Kathryn K. Harris,
  • Linda B. Bloom

摘要

The Escherichia coli helicase, YoaA, and DNA polymerase III subunit, χ, form a complex (YoaA-χ) that promotes tolerance to the DNA chain-terminator 3ˈ-azidothymidine (AZT). Single-stranded DNA binding protein (SSB), which accumulates at stalled replication forks, also contributes to AZT tolerance through interactions with χ. Here we show that in vitro, χ mediates interactions between YoaA and SSB that modulate helicase activity in a substrate-specific manner with little effect on overhang DNA but inhibiting unwinding of forked DNA. SSB similarly affects the activity of the YoaA paralog, DinG. Single-molecule experiments show that SSB translocates with YoaA-χ, increasing both the lifetime and frequency of SSB binding events. Mutational analyses show that χ binds at the back of YoaA relative to the direction of translocation supporting a model in which YoaA-χ pulls SSB along DNA as it translocates. To our knowledge, this is the first demonstration of a mechanoenzyme pulling SSB along ssDNA.