Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli.
BACKGROUND
recD, located between recB and argA, encodes the smallest polypeptide (60 kDa) of the heterotrimeric enzyme RecBCD in Escherichia coli. RecD is a 5'-3' helicase and is required for the nuclease activity of RecBCD and for tight binding to dsDNA ends. Here, we have tested the hypothesis that RecD regulates the structure and activities of RecBCD, including RecA loading.


RESULTS
To characterize its regulatory functions, recD was genetically fused to recB through deletion and substitution mutations. The recB-recD fusion led to a decreased amount of the heterotrimer. Both fusion mutants proved to be recombination proficient, viable and resistant to DNA damaging agents, and to have DNA unwinding, ATP-dependent dsDNA exonuclease and Chi genetic activities.


CONCLUSIONS
Our findings suggest that the recB-recD fusion may form a RecBD fusion protein and therefore affect RecD assembly, but this does not change the three-dimensional structure of the heterotrimer.
Biochemistry Department, Hacettepe University Medical School, 06100 Sihhiye, Ankara, Turkey. oytun@hacettepe.edu.tr
Oytun
O
Portakal
Pakize
P
Doğan
D000483:Alleles; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006238:Haploidy; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli