Genetic dissection of the biochemical activities of RecBCD enzyme.
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Abstract RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation. The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites. We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme. One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested. The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination. Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination. The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.Keywords:
RecBCD
The initial steps of double-stranded break (DSB) repair by homologous recombination mediated by the 5′–3′ exonuclease/annealing protein pairs, RecE/RecT and Redα/Redβ, were analyzed. Recombination was RecA-independent and required the expression of both components of an orthologous pair, even when the need for exonuclease activity was removed by use of preresected substrates. The required orthologous function correlated with a specific protein–protein interaction, and recombination was favored by overexpression of the annealing protein with respect to the exonuclease. The need for both components of an orthologous pair was observed regardless of whether recombination proceeded via a single-strand annealing or a putative strand invasion mechanism. The DSB repair reactions studied here are reminiscent of the RecBCD/RecA reaction and suggest a general mechanism that is likely to be relevant to other systems, including RAD52 mediated recombination.
RecBCD
RAD52
FLP-FRT recombination
Non-homologous end joining
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RecBCD
Nuclease
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Interaction with χ affects the helicase activity of RecBCD enzyme. Recognition of χ causes the enzyme to pause briefly at χ and to resume translocation after the χ site, but at a rate that is reduced by approximately twofold. In response to χ the RecBCD enzyme accomplishes both tasks essential for initiation of homologous recombination: (i) it recesses the double-strand break (DSB) to produce an ssDNA-tailed duplex DNA with χ at its terminus, and (ii) it catalyzes formation of the RecA nucleoprotein filament on the ssDNA produced. Interestingly, the efficiency of conjugational and transductional recombination by the RecF pathway in the recBC sbcBC cells is similar to that of the RecBCD pathway in wild-type cells, showing that the machinery of this pathway can be as productive as that of the RecBCD pathway. The loading of RecA protein is an essential aspect of recombination in the RecBCD pathway. On the other hand, recB recF double mutants are deficient in recombination between chromosomal direct repeats, suggesting that both RecBCD and RecF pathways play major roles in recombination. Homologous recombination can be initiated at either DSBs or single-strand DNA gap (SSG) in duplex DNA. Two major pathways are responsible for homologous recombination in wild-type E. coli: The RecBCD pathway is specific for the recombinational repair of DSBs, and in the wild-type cells, the RecF pathway is primarily used for recombination that initiates at SSGs.
RecBCD
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Non-allelic homologous recombination
FLP-FRT recombination
Site-specific recombination
Non-homologous end joining
Transposition (logic)
Branch migration
Ectopic recombination
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Non-allelic homologous recombination
Ectopic recombination
FLP-FRT recombination
Mitotic crossover
Transposition (logic)
Non-homologous end joining
Gene conversion
DNA Transposable Elements
Site-specific recombination
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Title of dissertation: Rapid kinetics study of the nuclease activity of RecBCD enzyme from Escherichia coli Archana Ghatak, Doctor of Philosophy, 2006 Dissertation directed by: Prof. Douglas A. Julin Department of Chemistry and Biochemistry RecBCD enzyme from Escherichia coli is involved in homologous recombination and repair of bacterial DNA, and in defending the cell against foreign DNA. This enzyme has multiple functions – it is an ATP-stimulated ssDNA endonuclease, an ATP-dependent ssDNA and dsDNA exonuclease and a DNA helicase. Here we investigated the kinetics of the exonuclease reaction of RecBCD. We purified the enzyme from E. coli by using conventional adsorption chromatography. The exonuclease function was studied using a rapid quench-flow (RQF) instrument designed by Kintek Corporation. Short single-stranded DNA oligomers were labeled with P ATP at the 5′ end and used as substrates. Our goal was to study the reaction products as the enzyme travels along the DNA. From earlier studies it is known that the enzyme binds at the end of a doublestranded DNA, unwinds and cleaves the DNA as it moves. This enzymatic movement can be very fast, in the range of 500−1000 base-pairs per second. We carried out exonuclease reactions using the RQF instrument for time periods ranging from 10 msec to 2 sec. The time courses of appearance and change of the amounts of the labeled products displayed in sequencing gels were analyzed using a Phosphorimager and associated software. A kinetic model for the enzyme was derived by analyzing the time course data with Kinteksim, a simulation program. We were able to identify the reaction products of the single turnover exonuclease reactions. For the first time we were able to show that the enzyme does not cleave a DNA substrate at every nucleotide even in conditions where it acted as a potent exonuclease. Rather, our results suggested that the enzyme moves along the DNA and cleaves in steps of 2-4 nucleotides. The enzyme concentration used in our study was almost 10 times more than the DNA concentration in order to make sure that all DNA molecules were bound to enzyme. Still not all DNA molecules participated in the nuclease reaction in the single turnover reaction time period, suggesting that some of the DNA enzyme complexes were inactive with respect to the nuclease activity. The rate of translocation of the enzyme along the DNA varied with the change of concentration of ATP in the reaction. However, the products were the same at several ATP concentrations. Our results indicate the presence of at least two ATP dependent steps after the reaction is started and before the first nuclease product appears. These steps are proposed to represent the translocation of the DNA end from the binding site to the nuclease active site. Also, our results suggest that the rate of translocation at each ATP concentration is similar to the unwinding rate of a double stranded DNA at the same ATP concentration [determined by Lucius, A. L. & Lohman, T. M. (2004) J Mol Biol 339, 751-771.] Rapid kinetics study of the nuclease activity of RecBCD enzyme from Escherichia coli
RecBCD
Nuclease
Enzyme Kinetics
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RecBCD
DNA polymerase I
DNA polymerase II
Klenow fragment
Exonuclease III
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Homologous recombination is a widely conserved process by which identical sequences of DNA cross over. This process is essential during cell division and increases genetic diversity. Homologous recombination is also an important mechanism in repairing DNA damage. In order for recombination to occur the cell must find a sequence of DNA identical to the one it must replace. Homologous recombination is carried out by and affected by a number of genes. RecA is the main factor in involved in homologous recombination as well as the DNA repair system. RecA loading onto the DNA is mediated by two different pathways, RecBCD and RecFOR. RecBCD recognizes double stranded break sand RecFOR pathway preferentially identifies single stranded gaps in DNA. Along with RecA, RecN plays a vital role in homologous recombination as it tethers DNA strands together during recombination. Two other vital genes involved in homologous recombination are RecG and RuvC, both which play important roles in resolving the Holliday junctions formed during recombination. There are also various mutagenic drugs that have an effect on DNA repair and homologous recombination mechanisms, such as AZT, 5- Azacytidine, Formaldehyde, and Ciprofloxacin. Using a lacZ reporter system and blue white screening we have developed a way in which to study homologous recombination at varying distances. Two chromosomal constructs have been created; one where a recipient LacZ has had a 5bp active site gene sequence deleted, this deletion creates an inactive LacZ protein and a donor sequence containing this 5bp deletion has been inserted 24minutes apart, at the attTn7 with 250bp of homology on either side of this 5bp active site. The second construct involves the complete deletion of the LacZ gene from its natural locus, and both the recipient, LacZ with the 5bp active site deletion, and donor sequences, the 5bp active site with 250bp homology on either side, have been inserted at the attTn7 site on the chromosome. These two constructs allow for the study of distance on homologous recombination both in regard to the genes involved in recombination that have been deleted, as well as to the portion of these constructs that were exposed to the various mutagenic drugs.
Non-allelic homologous recombination
RecBCD
Non-homologous end joining
Branch migration
FLP-FRT recombination
Homology directed repair
Site-specific recombination
In vitro recombination
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FLP-FRT recombination
Ectopic recombination
Non-homologous end joining
Non-allelic homologous recombination
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RecBCD
FLP-FRT recombination
Site-specific recombination
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Citations (27)