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The crystallographic structure of the RecBCD enzyme (PDB 1W36).[1] 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.

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, endonucleases, oxidative damage and a host of other factors.[2] 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.[1]

Contents

Structure

The enzyme complex is composed of three different subunits called RecB, RecC, and RecD and hence the complex is named RecBCD. Each subunit is encoded by a separate gene:

gene chain protein function
RecB beta P08394 3'-5' helicase, nuclease
RecC gamma P07648 recognizes χ (crossover hotspot instigator)
RecD alpha P04993 5'-3' helicase

Function

Beginning of the RecBCD pathway of homologous recombination.

Both the alpha (RecD) and beta (RecB) subunits are helicases, i.e. energy-dependent molecular motors that unwind double-stranded DNA or RNA. The beta subunit in addition has a nuclease function. Finally the gamma subunit (RecC) recognizes a specific sequence in DNA known as the chi site (also written with the Greek letter χ). In E. coli, in which chi sites are best studied, the sequence is 5'-GCTGGTGG-3'.

RecBCD is unusual amongst helicases because of its ability to recognize the chi DNA sequence. After it initiates unwinding, RecBCD cleaves both DNA strands endolytically, although the 5' tail is cleaved less often. When RecBCD encounters a chi site on this strand as it is unwinding DNA, it pauses and the digestion of the 3' tail ceases.[3] When RecBCD resumes unwinding, it now cleaves the opposite strand (i.e., the 5' tail). As a consequence, the 3' strand remains intact downstream of chi. The RecA protein is then loaded onto the 3' tail and RecBCD dissociates from the DNA. In a final step, RecA initiates strand invasion to continue the homologous recombination-based repair of the double stranded DNA break.

Application

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.[4]

References

  1. ^ a b Singleton MR, Dillingham MS, Gaudier M, Kowalczykowski SC, Wigley DB (November 2004). "Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks". Nature 432 (7014): 187–93. doi:10.1038/nature02988. PMID 15538360.  
  2. ^ Spies M, Kowalczykowski SC (2003). "Homologous recombination by RecBCD and RecF pathways". in Higgins P. Bacterial Chromosomes. Washington, D.C: ASM Press. pp. 389–403. ISBN 1-55581-232-5.  
  3. ^ Spies M, Amitani I, Baskin RJ, Kowalczykowski SC (November 2007). "RecBCD enzyme switches lead motor subunits in response to chi recognition". Cell 131 (4): 694–705. doi:10.1016/j.cell.2007.09.023. PMID 18022364.  
  4. ^ Bianco PR, Brewer LR, Corzett M, Balhorn R, Yeh Y, Kowalczykowski SC, Baskin RJ (January 2001). "Processive translocation and DNA unwinding by individual RecBCD enzyme molecules". Nature 409 (6818): 374–8. doi:10.1038/35053131. PMID 11201750.  

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