View source for RecBCD

{{Short description|Family of protein complexes in bacteria}}
{{Infobox enzyme
| Name       = Exodeoxyribonuclease V
| EC_number  = 3.1.11.5
| CAS_number = 37350-26-8
| GO_code    =
| image      = RecBCD 1W36.png
| width      = 
| caption    = '''Figure 1''' The crystallographic structure of the RecBCD enzyme ({{PDB|1W36}}).<ref name="pmid15538360"/> The RecB, RecC, and RecD subunits of the enzyme are colored cyan, green, and magenta respectively while the partially unwound DNA helix to which the enzyme is bound is colored brown. 
}}
'''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]] of [[Escherichia coli|''E. coli'']] that initiates [[recombinational repair]] from potentially lethal [[DNA repair#Double-strand breaks|double strand breaks]] in [[DNA]] which may result from [[ionizing radiation]], replication errors, [[endonuclease]]s, oxidative damage, and a host of other factors.<ref name=Smith2012>{{cite journal|last=Smith|first=GR|title=How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View|journal=Microbiol Mol Biol Rev|date=June 2012|volume=76|issue=2|pages=217–28|doi=10.1128/MMBR.05026-11|pmid=22688812|pmc=3372252}}</ref><ref name="isbn1-55581-232-5">{{cite book |vauthors=Spies M, Kowalczykowski SC | editor = Higgins P | title = Bacterial Chromosomes | publisher = ASM Press | location = Washington, D.C. | year = 2003 | pages = 389–403 | chapter = Homologous recombination by RecBCD and RecF pathways | isbn = 1-55581-232-5 }}</ref> 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.<ref name="pmid15538360">{{cite journal |vauthors=Singleton MR, Dillingham MS, Gaudier M, Kowalczykowski SC, Wigley DB | title = Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks | journal = Nature | volume = 432 | issue = 7014 | pages = 187–93 |date=November 2004 | pmid = 15538360 | doi = 10.1038/nature02988 | bibcode = 2004Natur.432..187S | s2cid = 2916995 }}</ref> 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,<ref name="pmid3526335">{{cite journal |vauthors=Amundsen SK, Taylor AF, Chaudhury AM, Smith GR | title = ''recD'': the gene for an essential third subunit of exonuclease V. | journal = Proc Natl Acad Sci U S A | volume = 83 | issue = 15 | pages = 5558–62 |date=August 1986 | pmid = 3526335 | pmc = 386327 | doi = 10.1073/pnas.83.15.5558| bibcode = 1986PNAS...83.5558A | doi-access = free }}</ref> the enzyme was known as “RecBC.”  Each subunit is encoded by a separate gene:

{| class="wikitable"
|-
! gene
! chain
! protein
! function
|-
| RecB
| β
| {{Uniprot|P08394}}
| 3'-5' helicase, nuclease
|-
| RecC
| γ
| {{Uniprot|P07648}}
| Recognizes Chi (''c''rossover ''h''otspot ''i''nstigator)
|-
| RecD
| α
| {{Uniprot|P04993}}
| 5'-3' helicase
|}

==Function==
{{Main | Homologous_recombination#RecBCD_pathway | l1=Homologous recombination: RecBCD pathway}}
[[File:Chi Recombination Model for Wikipedia.tif|thumb|left| '''Figure 2'''  RecBCD pathway of homologous recombination where ATP is in excess.]]

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.<ref name="pmid9448271">{{cite journal |vauthors=Yu M, Souaya J, Julin DA | title = The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from ''Escherichia coli'' | journal = Proc Natl Acad Sci U S A | volume = 95 | issue = 3 | pages = 981–6 |date=February 1998 | pmid = 9448271 | pmc = 18645 | doi = 10.1073/pnas.95.3.981| bibcode = 1998PNAS...95..981Y | doi-access = free }}</ref>  Finally, RecBCD enzyme (perhaps the RecC subunit) recognizes a specific sequence in DNA, [[5']]-GCTGGTGG-[[3']], known as [[Chi site|Chi]] (sometimes designated with the Greek letter χ).

RecBCD is unusual amongst helicases because it has two helicases that travel with different rates<ref name="pmid12815437">{{cite journal |vauthors=Taylor AF, Smith GR | title = RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity | journal = Nature | volume = 423 | issue = 6942 | pages = 889–93 |date=June 2003 | pmid = 12815437 | doi = 10.1038/nature01674 | bibcode = 2003Natur.423..889T | s2cid = 4302346 }}</ref> and because it can recognize and be altered by the Chi DNA sequence.<ref name="pmid1535156">{{cite journal |vauthors=Taylor AF, Smith GR | title = RecBCD enzyme is altered upon cutting DNA at a Chi recombination hotspot | journal = Proc Natl Acad Sci U S A | volume = 89 | issue = 12 | pages = 5226–30 |date=June 1992 | pmid = 1535156 | pmc = 49264 | doi =  10.1073/pnas.89.12.5226| bibcode = 1992PNAS...89.5226T | doi-access = free }}</ref><ref name="pmid18079176">{{cite journal |vauthors=Amundsen SK, Taylor AF, Reddy M, Smith GR | title = Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots | journal = Genes Dev | volume = 21 | issue = 24 | pages = 3296–307 |date=December 2007 | pmid = 18079176 | pmc = 2113030 | doi = 10.1101/gad.1605807 }}</ref>  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.<ref name="pmid6256081">{{cite journal |vauthors=Taylor A, Smith GR | title = Unwinding and rewinding of DNA by the RecBC enzyme | journal = Cell | volume = 22 | issue = 2 Pt 2| pages = 447–57 |date=November 1980 | pmid = 6256081 | doi = 10.1016/0092-8674(80)90355-4 | s2cid = 22123298 }}</ref>  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.”
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== Mechanism of action ==
[[Image:HR RecBCD RecA.svg|250px|thumb | '''Figure 3''' Beginning of the RecBCD pathway of homologous recombination where Mg<sup>2+</sup> is in excess.]]

During unwinding the nuclease in RecB can act in different ways depending on the reaction conditions, notably the ratio of the concentrations of Mg<sup>2+</sup> 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).<ref>{{Cite journal|last1=Ponticelli|first1=A. S.|last2=Schultz|first2=D. W.|last3=Taylor|first3=A. F.|last4=Smith|first4=G. R.|date=May 1985|title=Chi-dependent DNA strand cleavage by RecBC enzyme|journal=Cell|volume=41|issue=1|pages=145–151|doi=10.1016/0092-8674(85)90069-8|pmid=3888404|s2cid=38361329}}</ref><ref name="pmid3888405">{{cite journal |vauthors=Taylor AF, Schultz DW, Ponticelli AS, Smith GR | title = RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting | journal = Cell | volume = 41 | issue = 1 | pages = 153–63 |date=May 1985 | pmid = 3888405 | doi =  10.1016/0092-8674(85)90070-4| s2cid = 8551482 }}</ref>  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.<ref name="pmid9230304">{{cite journal |vauthors=Anderson DG, Kowalczykowski SC | title = The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a Chi-regulated manner | journal = Cell | volume = 90 | issue = 1 | pages = 77–86 |date=July 1997 | pmid = 9230304 | doi =  10.1016/S0092-8674(00)80315-3| doi-access = free }}</ref> When RecBCD reaches the end of the DNA, all three subunits disassemble and the enzyme remains inactive for an hour or more;<ref name="pmid10197988">{{cite journal |vauthors=Taylor AF, Smith GR | title = Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits | journal = Genes Dev. | volume = 13 | issue = 7 | pages = 890–900 |date=April 1999 | pmid = 10197988 | pmc = 316601 | doi =  10.1101/gad.13.7.890}}</ref> a RecBCD molecule that acted at Chi does not attack another DNA molecule.  '''(2)''' If Mg<sup>2+</sup> ions are in excess, RecBCD cleaves both DNA strands endonucleolytically, although the 5' tail is cleaved less often (Figure 3).<ref name="pmid8384931">{{cite journal |vauthors=Dixon DA, Kowalczykowski SC | title = The recombination hotspot Chi is a regulatory sequence that acts by attenuating the nuclease activity of the ''E. coli'' RecBCD enzyme | journal = Cell | volume = 73 | issue = 1 | pages = 87–96 |date=April 1993 | pmid = 8384931 | doi =  10.1016/0092-8674(93)90162-J| s2cid = 19718817 }}</ref>  When RecBCD encounters a Chi site on the 3' ended strand, unwinding pauses and digestion of the 3' tail is reduced.<ref name="pmid18022364">{{cite journal |vauthors=Spies M, Amitani I, Baskin RJ, Kowalczykowski SC | title = RecBCD enzyme switches lead motor subunits in response to Chi recognition | journal = Cell | volume = 131 | issue = 4 | pages = 694–705 |date=November 2007 | pmid = 18022364 | pmc =  2151923| doi = 10.1016/j.cell.2007.09.023 }}</ref>  When RecBCD resumes unwinding, it now cleaves the opposite strand (''i.e.'', the 5' tail)<ref name="pmid7592661">{{cite journal |vauthors=Taylor AF, Smith GR | title = Strand specificity of nicking of DNA at Chi sites by RecBCD enzyme. Modulation by ATP and magnesium levels | journal = J Biol Chem | volume = 270 | issue = 41 | pages = 24459–67 |date=October 1995 | pmid = 7592661 | doi =  10.1074/jbc.270.41.24459| doi-access = free }}</ref><ref name="pmid9119222">{{cite journal |vauthors=Anderson DG, Kowalczykowski SC | title = The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme | journal = Genes Dev. | volume = 11 | issue = 5 | pages = 571–81 |date=March 1997 | pmid = 9119222 | doi =  10.1101/gad.11.5.571| doi-access = free }}</ref> and loads RecA protein onto the 3’-ended strand.<ref name="pmid9230304"/>  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.<ref name="Smith2012" />  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<sup>2+</sup> in excess [PMIDs 27401752,  27330137]. RecBCD mutants lacking detectable exonuclease activity retain high Chi hotspot activity in cells and nicking at Chi outside cells.<ref name="pmid17110484">{{cite journal |vauthors=Amundsen SK, Smith GR | title = Chi hotspot activity in ''Escherichia coli'' without RecBCD exonuclease activity: implications for the mechanism of recombination | journal = Genetics | volume = 175 | issue = 1 | pages = 41–54 |date=January 2007 | pmid = 17110484 | pmc = 1774988 | doi =  10.1534/genetics.106.065524}}</ref>  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.<ref name="pmid7541534">{{cite journal |vauthors=Köppen A, Krobitsch S, Thoms B, Wackernagel W | title = Interaction with the recombination hot spot Chi in vivo converts the RecBCD enzyme of ''Escherichia coli'' into a Chi-independent recombinase by inactivation of the RecD subunit | journal = Proc Natl Acad Sci U S A | volume = 92 | issue = 14 | pages = 6249–53 |date=July 1995 | pmid = 7541534 | pmc = 41495 | doi =  10.1073/pnas.92.14.6249| bibcode = 1995PNAS...92.6249K | doi-access = free }}</ref><ref name="pmid7603978">{{cite journal |vauthors=Myers RS, Kuzminov A, Stahl FW | title = The recombination hot spot Chi activates RecBCD recombination by converting ''Escherichia coli'' to a recD mutant phenocopy | journal = Proc Natl Acad Sci U S A | volume = 92 | issue = 14 | pages = 6244–8 |date=July 1995 | pmid = 7603978 | pmc = 41494 | doi =  10.1073/pnas.92.14.6244| bibcode = 1995PNAS...92.6244M | doi-access = free }}</ref>

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.<ref name="pmid9230304"/>  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.

==RecD1 and RecD2==

RecD enzymes are divided into two groups RecD1 (known as RecD) and RecD2.<ref name = Ramos2022>{{cite journal |vauthors=Ramos C, Hernández-Tamayo R, López-Sanz M, Carrasco B, Serrano E, Alonso JC, Graumann PL, Ayora S |title=The RecD2 helicase balances RecA activities |journal=Nucleic Acids Res |volume=50 |issue=6 |pages=3432–3444 |date=April 2022 |pmid=35234892 |pmc=8989531 |doi=10.1093/nar/gkac131 |url=}}</ref>  Many organisms have a ''recD'' gene even though the other members of a recBCD complex, i. e. rec B and recC, are not present.<ref name = Montague2009>{{cite journal |vauthors=Montague M, Barnes C, Smith HO, Chuang RY, Vashee S |title=The evolution of RecD outside of the RecBCD complex |journal=J Mol Evol |volume=69 |issue=4 |pages=360–71 |date=October 2009 |pmid=19841849 |doi=10.1007/s00239-009-9290-x |bibcode=2009JMolE..69..360M |url=}}</ref>  For instance, the bacterium ''[[Deinococcus radiodurans]]'', that has an extraordinary [[DNA repair]] capability, is an example of an organism that does not possess a ''recB'' or ''recC'' gene, and yet does have a ''recD'' gene.<ref name = Montague2009/>  In the bacterium ''[[Escherichia coli]]'', RecD protein is part of the well studied RecBCD complex that is necessary for [[genetic recombination|recombinational DNA repair]] (as described above).  In the bacterium ''[[Bacillus subtilis]]'', RecD2 protein has a role as a modulator of replication restart and also a modulator of the [[RecA]] recombinase.<ref name = Ramos2022/>  RecD2 may inhibit unwanted recombination events when replication forks are stalled, and also may have a role in displacing RecA protein from recombination intermediates in order to permit advance of the replication fork.<ref name = Ramos2022/>

== Applications ==
RecBCD is a model enzyme for the use of [[single-molecule FRET|single molecule fluorescence]] as an experimental technique used to better understand the function of protein-DNA interactions.<ref name="pmid11201750">{{cite journal |vauthors=Bianco PR, Brewer LR, Corzett M, Balhorn R, Yeh Y, Kowalczykowski SC, Baskin RJ | title = Processive translocation and DNA unwinding by individual RecBCD enzyme molecules | journal = Nature | volume = 409 | issue = 6818 | pages = 374–8 |date=January 2001 | pmid = 11201750 | doi = 10.1038/35053131 | bibcode = 2001Natur.409..374B | s2cid = 4399125 }}</ref>  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.
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== References ==
{{Reflist|35em}}

== External links ==
* {{MeshName|Exodeoxyribonuclease+V}}
* {{MeshName|Exodeoxyribonuclease+V,+E+coli}}
* {{EC number|3.1.11.5}}

{{Escherichia coli}}
{{Esterases}}
{{Enzymes}}
{{DNA repair}}
{{Portal bar|Biology|border=no}}

[[Category:EC 3.1.11]]
[[Category:Escherichia coli genes]]
[[Category:DNA repair]]