In vivo studies of the Escherichia coli RecB polypeptide lacking its nuclease center.
In vitro, RecB1-929, the truncated Escherichia coli RecB polypeptide, comprising the N-terminal (helicase) domain of RecB, can combine with RecC and RecD subunits of RecBCD enzyme. The resulting RecB1-929CD heterotrimer is a potent helicase; due to the loss of the nuclease center of RecB, it is devoid of DNase activities. By making use of the RecB1-929-producing plasmid pMY100, the in vivo behavior of this truncated polypeptide was studied. The following observations were made. (i) Large amounts of RecB1-929 in the pulse-heated lambdacI857gam+ lysogens prevented the growth of a gene 2 mutant of bacteriophage T4. It may be inferred that lambda-Gam protein, which otherwise inhibits RecBCD DNase and thus permits the growth of this phage, is bound by the helicase domain of RecB. (ii) The simultaneous presence of RecB1-929, RecC, and RecD did not restore recombination proficiency and ultraviolet resistance of recB cells. (iii) The presence of RecB1-929 did not alter recombination and repair processes in wild-type (recBCD+) cells. Even excessively large amounts of this truncated polypeptide did not reduce degradation of chromosomal DNA damaged by y-rays. It may be inferred that under in vivo conditions, the 30-kDa domain of RecB is essential for assembly of the RecBCD enzyme and/or for holding its three subunits together.
Salaj-Smic
E
E
Department of Molecular Genetics, Ruder Bosković Institute, Zagreb, Croatia. salaj@rudjer.irb.hr
Dermić
D
D
Brcić-Kostić
K
K
Cajo
G C
GC
Trgovcević
E
E
D010582:Bacteriophage lambda / Q000235:genetics / Q000502:physiology; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects / Q000821:virology; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005720:Gamma Rays; D011995:Recombination, Genetic; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000378:metabolism
D029968:Escherichia coli Proteins; D014764:Viral Proteins; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli