RecBCD
Encyclopedia
RecBCD, also known as Exonuclease V, is an enzyme
of the E. coli
bacterium that initiates recombinational repair from potentially lethal double strand breaks in DNA
which may result from ionizing radiation, replication errors, endonuclease
s, oxidative damage, and a host of other factors. The RecBCD enzyme is both a helicase
that unwinds, or separates the strands of, DNA and a nuclease
that makes single-stranded nicks in DNA.
(sometimes designated with the Greek letter χ).
RecBCD is unusual amongst helicases because it has two helicases that travel with different rates and because it can recognize and be altered by the Chi DNA sequence. RecBCD avidly binds an end of linear double-stranded (ds) DNA. The RecD helicase travels on the strand with a 5' end at which the enzyme initiates unwinding, and RecB on the strand with a 3' end. RecB is slower than RecD, so that a single-stranded (ss) DNA loop accumulates ahead of RecB (Figure 2). This produces DNA structures with two ss tails (a shorter 3’ ended tail and a longer 5’ ended tail) and one ss loop (on the 3' ended strand) observed by electron microscopy. The ss tails can anneal to produce a second ss loop complementary to the first one; such twin-loop structures were initially referred to as “rabbit ears.”
Although neither reaction has been verified by analysis of intracellular DNA, due to their transient nature, genetic evidence indicates that the first reaction more nearly mimics that in cells. For example, RecBCD mutants lacking detectable exonuclease activity retain high Chi hotspot activity in cells and nicking at Chi outside cells. A Chi site on one DNA molecule in cells reduces or eliminates Chi activity on another DNA, perhaps reflecting the Chi-dependent disassembly of RecBCD observed in vitro under conditions of excess ATP and nicking of DNA at Chi.
Under both reaction conditions, the 3' strand remains intact downstream of Chi. The RecA
protein is then actively loaded onto the 3' tail by RecBCD. At some undetermined point RecBCD dissociates from the DNA, although RecBCD can unwind at least 60 kb of DNA without falling off. RecA initiates exchange of the DNA strand to which it is bound with the identical, or nearly identical, strand in an intact DNA duplex; this strand exchange generates a joint DNA molecule, such as a D-loop (Figure 2). The joint DNA molecule is thought to be resolved either by replication primed by the invading 3’ ended strand containing Chi or by cleavage of the D-loop and formation of a Holliday junction. The Holliday junction
can be resolved into linear DNA by the RuvABC
complex or dissociated by the RecG protein. Each of these events can generate intact DNA with new combinations of genetic markers by which the parental DNAs may differ. This process, homologous recombination
, completes the repair of the double-stranded DNA break.
as an experimental technique used to better understand the function of protein-DNA interactions. The enzyme is also useful in removing linear DNA, either single- or double-stranded, from preparations of circular double-stranded DNA, since it requires a DNA end for activity.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
of the E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
bacterium that initiates recombinational repair from potentially lethal double strand breaks in DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
which may result from ionizing radiation, replication errors, endonuclease
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
s, oxidative damage, and a host of other factors. The RecBCD enzyme is both a helicase
Helicase
Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.-Function:Many cellular processes Helicases are a...
that unwinds, or separates the strands of, DNA and a nuclease
Nuclease
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase"....
that makes single-stranded nicks in DNA.
Structure
The enzyme complex is composed of three different subunits called RecB, RecC, and RecD and hence the complex is named RecBCD (Figure 1). Before the discovery of the recD gene, the enzyme was known as “RecBC.” Each subunit is encoded by a separate gene:| gene | chain | protein | function |
|---|---|---|---|
| RecB | beta | 3'-5' helicase, nuclease | |
| RecC | gamma | Likely recognizes Chi (crossover hotspot instigator) | |
| RecD | alpha | 5'-3' helicase |
Function
Both the RecD and RecB subunits are helicases, i.e., energy-dependent molecular motors that unwind DNA (or RNA in the case of other proteins). The RecB subunit in addition has a nuclease function. Finally, RecBCD enzyme (perhaps the RecC subunit) recognizes a specific sequence in DNA, 5'-GCTGGTGG-3', known as ChiChi site
A Chi site or Chi sequence is a short stretch of DNA in the genome of a bacterium near which homologous recombination is unusually likely to occur. Chi sites serve as stimulators of DNA double-strand break repair in bacteria, which can arise from radiation or chemical treatments, or result from...
(sometimes designated with the Greek letter χ).
RecBCD is unusual amongst helicases because it has two helicases that travel with different rates and because it can recognize and be altered by the Chi DNA sequence. RecBCD avidly binds an end of linear double-stranded (ds) DNA. The RecD helicase travels on the strand with a 5' end at which the enzyme initiates unwinding, and RecB on the strand with a 3' end. RecB is slower than RecD, so that a single-stranded (ss) DNA loop accumulates ahead of RecB (Figure 2). This produces DNA structures with two ss tails (a shorter 3’ ended tail and a longer 5’ ended tail) and one ss loop (on the 3' ended strand) observed by electron microscopy. The ss tails can anneal to produce a second ss loop complementary to the first one; such twin-loop structures were initially referred to as “rabbit ears.”
Mechanism of action
During unwinding the nuclease in RecB can act in different ways depending on the reaction conditions, notably the ratio of the concentrations of Mg2+ ions and ATP. (1) If ATP is in excess, the enzyme simply nicks the strand with Chi (the strand with the initial 3' end) (Figure 2). Unwinding continues and produces a 3' ss tail with Chi near its terminus. This tail can be bound by RecA protein, which promotes strand exchange with an intact homologous DNA duplex. When RecBCD reaches the end of the DNA, all three subunits disassemble and the enzyme remains inactive for an hour or more; a RecBCD molecule that acted at Chi does not attack another DNA molecule. (2) If Mg2+ ions are in excess, RecBCD cleaves both DNA strands endonucleolytically, although the 5' tail is cleaved less often (Figure 3). When RecBCD encounters a Chi site on the 3' ended strand, unwinding pauses and digestion of the 3' tail is reduced. When RecBCD resumes unwinding, it now cleaves the opposite strand (i.e., the 5' tail) and loads RecA protein onto the 3’-ended strand. After completing reaction on one DNA molecule, the enzyme quickly attacks a second DNA, on which the same reactions occur as on the first DNA.Although neither reaction has been verified by analysis of intracellular DNA, due to their transient nature, genetic evidence indicates that the first reaction more nearly mimics that in cells. For example, RecBCD mutants lacking detectable exonuclease activity retain high Chi hotspot activity in cells and nicking at Chi outside cells. A Chi site on one DNA molecule in cells reduces or eliminates Chi activity on another DNA, perhaps reflecting the Chi-dependent disassembly of RecBCD observed in vitro under conditions of excess ATP and nicking of DNA at Chi.
Under both reaction conditions, the 3' strand remains intact downstream of Chi. The RecA
RecA
RecA is a 38 kilodalton Escherichia coli protein essential for the repair and maintenance of DNA. RecA has a structural and functional homolog in every species in which it has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins...
protein is then actively loaded onto the 3' tail by RecBCD. At some undetermined point RecBCD dissociates from the DNA, although RecBCD can unwind at least 60 kb of DNA without falling off. RecA initiates exchange of the DNA strand to which it is bound with the identical, or nearly identical, strand in an intact DNA duplex; this strand exchange generates a joint DNA molecule, such as a D-loop (Figure 2). The joint DNA molecule is thought to be resolved either by replication primed by the invading 3’ ended strand containing Chi or by cleavage of the D-loop and formation of a Holliday junction. The Holliday junction
Holliday junction
A Holliday junction is a mobile junction between four strands of DNA. The structure is named after Robin Holliday, who proposed it in 1964 to account for a particular type of exchange of genetic information he observed in yeast known as homologous recombination...
can be resolved into linear DNA by the RuvABC
RuvABC
RuvABC is a complex of three proteins that mediate branch migration and resolve the Holliday junction created during homologous recombination in bacteria. As such, RuvABC is critical to bacterial DNA repair....
complex or dissociated by the RecG protein. Each of these events can generate intact DNA with new combinations of genetic markers by which the parental DNAs may differ. This process, homologous recombination
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
, completes the repair of the double-stranded DNA break.
Applications
RecBCD is a model enzyme for the use of single molecule fluorescenceSingle-molecule FRET
Single-molecule Fluorescence Resonance Energy Transfer is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a single donor and acceptor FRET pair are excited and detected...
as an experimental technique used to better understand the function of protein-DNA interactions. The enzyme is also useful in removing linear DNA, either single- or double-stranded, from preparations of circular double-stranded DNA, since it requires a DNA end for activity.