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

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Abstract
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.
Funding Information
  • U.S. Department of Health & Human Services | National Institutes of Health (R01GM140166, F31ES03465, ES034652)
  • National Science Foundation (MCB 1817869)

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