Effects of recJ, recQ, and recFOR Mutations on Recombination in Nuclease-Deficient recB recD Double Mutants of Escherichia coli

ABSTRACT The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5′→3′ exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5′→3′ exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5′→3′ exonuclease is an element of the RecF recombination machinery.

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The initiation and control of homologous recombination in escherichia coli

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1995.0003 ◽

1995 ◽

Vol 347 (1319) ◽

pp. 13-20 ◽

Cited By ~ 29

Keyword(s):

Escherichia Coli ◽

Low Frequency ◽

Nuclease Activity ◽

X Rays ◽

Single Stranded Dna ◽

Dna Break ◽

Dna Break Repair ◽

Break Repair ◽

Recbcd Enzyme ◽

Dna Ends

The chromosome of Escherichia coli recombines at low frequency when it is an intact circle but recombines at high frequency when it is broken, for example by X-rays, or when a linear DNA fragment is introduced into the cell during conjugation or transduction. The high recombinogenicity of double-strand (ds) DNA ends is attributable to RecBCD enzyme, which acts on ds DNA ends and is essential for recombination and ds DNA break repair. RecBCD enzyme initiates DNA unwinding at ds DNA ends, and its nuclease activity is controlled by Chi sites (5' G-C-T-G-G-T-G-G 3') in such a way that the enzyme produces a potent single-stranded DNA substrate for homologous pairing by RecA and single-stranded DNA binding proteins. We discuss a unifying model for recombination and ds DNA break repair, based upon the enzymic activities of these and other proteins and upon the behaviour of E. coli mutants altered in these proteins.

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RecFOR Function Is Required for DNA Repair and Recombination in a RecA Loading-Deficient recB Mutant of Escherichia coli

Genetics ◽

10.1093/genetics/163.2.485 ◽

2003 ◽

Vol 163 (2) ◽

pp. 485-494 ◽

Cited By ~ 1

Author(s):

Ivana Ivančić-Baće ◽

Petra Peharec ◽

Sunčana Moslavac ◽

Nikolina Škrobot ◽

Erika Salaj-Šmic† ◽

...

Keyword(s):

Escherichia Coli ◽

Reca Protein ◽

Nuclease Activity ◽

Filament Formation ◽

Single Stranded Dna ◽

Catalytic Center ◽

In Vivo Assays ◽

Recbcd Enzyme ◽

Contradictory Data

Abstract The RecA loading activity of the RecBCD enzyme, together with its helicase and 5′ → 3′ exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF– background the recB1080 mutant is recombination deficient, whereas in a recF+ genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D–) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5′ → 3′ exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.

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sbcB15 and ΔsbcB Mutations Activate Two Types of RecF Recombination Pathways in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00613-06 ◽

2006 ◽

Vol 188 (21) ◽

pp. 7562-7571 ◽

Cited By ~ 8

Author(s):

Ksenija Zahradka ◽

Sanela Šimić ◽

Maja Buljubašić ◽

Mirjana Petranović ◽

Damir Đermić ◽

...

Keyword(s):

Escherichia Coli ◽

Recq Helicase ◽

Dna Breaks ◽

Γ Irradiation ◽

E Coli ◽

Exonuclease I ◽

Double Stranded Dna ◽

Recombination Processes ◽

Recf Pathway ◽

Recombination Reactions

ABSTRACT Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and ΔsbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and ΔsbcB mutations on DNA repair after UV and γ irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than ΔsbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as ΔsbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC ΔsbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.

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