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

Abstract

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.

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

“…Loss of Uup or RadD function alone had no significant effects on their own with any DNA damaging agent (Figure 8A ). Loss of Uup and RadD together also had minimal effects except in the cases of Mitomycin C (MMC) and Trimethoprim (Trim) (increased sensitivity of the double deletion mutant has also been noted for Ciprofloxacin (Cipro) at levels higher than used here ( 33 )). These results are consistent with earlier observations and provide one rationale for why Uup and RadD were largely overlooked until recently.…”

“…Uup is a UvrA-like Class II ABC system that binds Holliday junctions. RadD and Uup help define at least two pathways for resolution of branched DNA intermediates during template switching in post replication gaps ( 33 ). Uup and RadD are responsible for the stabilization of tandem repeats that are susceptible to deletion ( 33 ).…”

“…A ΔrnhAΔrecG double mutant cannot be constructed, with no viability at any temperature with or without a dnaA(46) mutation. (c) Whereas a ΔrecGΔradD strain cannot grow, a ΔuupΔradD strain grows well in the absence of stress ( 33 ). (d) Eliminating Uup does not affect the sensitivity of a recG deletion to UV irradiation or Trimethoprim although it does suppress the ΔrecG sensitivity to a number of other agents.…”

“…RadD shares significant homology to the E. coli SF2 helicases RecQ and RecG. Previous work has shown RadD suppresses crossovers that can occur in postreplication gaps and can bind forked DNA structures ( 33 ). It is important for survival during tobramycin and ionizing radiation treatment.…”

ABC‐F translation factors: from antibiotic resistance to immune response

“…[33,61]. Uup is also implicated in DNA repair, function that also request DNA recombination like transposon excision [62].…”