UmuD and RecA* modulate the DNA-binding activity of DNA polymerase IV in Escherichia coli

Expression of DNA polymerase IV (pol IV) is increased in Escherichia coli cells suffering high levels of DNA damage. Despite the resulting increase in intracellular pol IV concentration, the binding of pol IV to the nucleoid does not significantly increase, indicating that pol IV activity is highly regulated in cells. Here, we use quantitative single-molecule imaging to investigate how UmuD and RecA, two binding partners of pol IV regulate its activity in live cells. We find that UmuD acts as a positive regulator of pol IV activity, promoting long-lived association of pol IV with the DNA. In contrast, its cleaved form which accumulates in DNA-damaged cells, UmuD?, acts as a negative regulator by inhibiting the binding of pol IV to DNA. In cells treated with the antibiotic ciprofloxacin, up to 40% of pol IV foci colocalise with RecA* nucleoprotein filaments. RecA(E38K), a hyperactive RecA mutant, recruits pol IV to the nucleoid, even in the absence of exogenous DNA damage. In vitro, RecA(E38K) forms RecA*-like structures competent of LexA cleavage, even on double-stranded DNA. Using surface plasmon resonance, we show that these RecA*-like filaments can recruit pol IV to double-stranded DNA, consistent with a physical interaction between RecA and pol IV. Together, the results show UmuD and RecA as modulators of DNA binding by pol IV and reveal new elements of a mutagenesis ‘switch’ that occurs in DNA damaged cells. Our findings strongly support the notion that repair synthesis during homologous recombination represents a major function of pol IV in cells.