Exodeoxyribonuclease V (EC 3.1.11.5, RecBCD, Exonuclease V, ''Escherichia coli'' exonuclease V, ''E. coli'' exonuclease V, gene recBC endoenzyme, RecBC deoxyribonuclease, gene recBC DNase, gene recBCD enzymes) is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
of
''E. coli'' that initiates
recombinational repair
Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may be ...
from potentially lethal
double strand breaks in
DNA which may result from
ionizing radiation, replication errors,
endonucleases, oxidative damage, and a host of other factors.
The RecBCD enzyme is both a
helicase
Helicases are a class of enzymes thought to be vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separatin ...
that unwinds, or separates the strands of DNA, and a
nuclease that makes single-stranded nicks in DNA.
It catalyses exonucleolytic cleavage (in the presence of ATP) in either 5′- to 3′- or 3′- to 5′-direction to yield 5′-phosphooligonucleotides.
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:
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
Chi
Chi or CHI may refer to:
Greek
*Chi (letter), the Greek letter (uppercase Χ, lowercase χ);
Chinese
*Chi (length), ''Chi'' (length) (尺), a traditional unit of length, about ⅓ meter
*Chi (mythology) (螭), a dragon
*Chi (surname) (池, pin ...
(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 Mg
2+ 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 Mg
2+ 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 the transient nature of reaction intermediates, genetic evidence indicates that the first reaction more nearly mimics that in cells.
For example, the activity of Chi is influenced by nucleotides to its 3' side, both in cells and in reactions with ATP in excess but not with Mg
2+ in excess
MIDs 27401752, 27330137 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 is a 38 kilodalton protein essential for the repair and maintenance of DNA. A RecA structural and functional homolog has been found in every species in which one has been seriously sought and serves as an archetype for this class of homolog ...
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
In molecular biology, a displacement loop or D-loop is a DNA structure where the two strands of a double-stranded DNA molecule are separated for a stretch and held apart by a third strand of DNA. An R-loop is similar to a D-loop, but in this ca ...
(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
RuvABC ( Recombination UV) 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.
RuvA and Ruv ...
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 is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may ...
, completes the repair of the double-stranded DNA break.
Applications
RecBCD is a model enzyme for the use of
single molecule fluorescence 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.
References
External links
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Molecular biology
EC 3.1.11
Escherichia coli genes