RadD is a RecA-dependent accessory protein that accelerates DNA strand exchange.

In rapidly growing cells, with recombinational DNA repair required often and a new replication fork passing every 20 min, the pace of RecA-mediated DNA strand exchange is potentially much too slow for bacterial DNA metabolism. The enigmatic RadD protein, a putative SF2 family helicase, exhibits no independent helicase activity on branched DNAs. Instead, RadD greatly accelerates RecA-mediated DNA strand exchange, functioning only when RecA protein is present. The RadD reaction requires the RadD ATPase activity, does not require an interaction with SSB, and may disassemble RecA filaments as it functions. We present RadD as a new class of enzyme, an accessory protein that accelerates DNA strand exchange, possibly with a helicase-like action, in a reaction that is entirely RecA-dependent. RadD is thus a DNA strand exchange (recombination) synergist whose primary function is to coordinate closely with and accelerate the DNA strand exchange reactions promoted by the RecA recombinase. Multiple observations indicate a uniquely close coordination of RadD with RecA function.

RecA-independent recombination: Dependence on the Escherichia coli RarA protein.

Most, but not all, homologous genetic recombination in bacteria is mediated by the RecA recombinase. The mechanistic origin of RecA-independent recombination has remained enigmatic. Here, we demonstrate that the RarA protein makes a major enzymatic contribution to RecA-independent recombination. In particular, RarA makes substantial contributions to intermolecular recombination and to recombination events involving relatively short (<200 bp) homologous sequences, where RecA-mediated recombination is inefficient. The effects are seen here in plasmid-based recombination assays and in vivo cloning processes. Vestigial levels of recombination remain even when both RecA and RarA are absent. Additional pathways for RecA-independent recombination, possibly mediated by helicases, are suppressed by exonucleases ExoI and RecJ. Translesion DNA polymerases may also contribute. Our results provide additional substance to a previous report of a functional overlap between RecA and RarA.

Resolving Toxic DNA repair intermediates in every E. coli replication cycle: critical roles for RecG, Uup and RadD.

DNA lesions or other barriers frequently compromise replisome progress. The SF2 helicase RecG is a key enzyme in the processing of postreplication gaps or regressed forks in Escherichia coli. A deletion of the recG gene renders cells highly sensitive to a range of DNA damaging agents. Here, we demonstrate that RecG function is at least partially complemented by another SF2 helicase, RadD. A ΔrecGΔradD double mutant exhibits an almost complete growth defect, even in the absence of stress. Suppressors appear quickly, primarily mutations that compromise priA helicase function or recA promoter mutations that reduce recA expression. Deletions of uup (encoding the UvrA-like ABC system Uup), recO, or recF also suppress the ΔrecGΔradD growth phenotype. RadD and RecG appear to avoid toxic situations in DNA metabolism, either resolving or preventing the appearance of DNA repair intermediates produced by RecA or RecA-independent template switching at stalled forks or postreplication gaps. Barriers to replisome progress that require intervention by RadD or RecG occur in virtually every replication cycle. The results highlight the importance of the RadD protein for general chromosome maintenance and repair. They also implicate Uup as a new modulator of RecG function.

Frequent template switching in postreplication gaps: suppression of deleterious consequences by the Escherichia coli Uup and RadD proteins.

When replication forks encounter template DNA lesions, the lesion is simply skipped in some cases. The resulting lesion-containing gap must be converted to duplex DNA to permit repair. Some gap filling occurs via template switching, a process that generates recombination-like branched DNA intermediates. The Escherichia coli Uup and RadD proteins function in different pathways to process the branched intermediates. Uup is a UvrA-like ABC family ATPase. RadD is a RecQ-like SF2 family ATPase. Loss of both functions uncovers frequent and RecA-independent deletion events in a plasmid-based assay. Elevated levels of crossing over and repeat expansions accompany these deletion events, indicating that many, if not most, of these events are associated with template switching in postreplication gaps as opposed to simple replication slippage. The deletion data underpin simulations indicating that multiple postreplication gaps may be generated per replication cycle. Both Uup and RadD bind to branched DNAs in vitro. RadD protein suppresses crossovers and Uup prevents nucleoid mis-segregation. Loss of Uup and RadD function increases sensitivity to ciprofloxacin. We present Uup and RadD as genomic guardians. These proteins govern two pathways for resolution of branched DNA intermediates such that potentially deleterious genome rearrangements arising from frequent template switching are averted.

Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response.

The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.