SSB protein controls RecBCD enzyme nuclease activity during unwinding: a new role for looped intermediates.
The RecBCD enzyme of Escherichia coli initiates homologous recombination by unwinding and simultaneously degrading DNA from a double-stranded DNA end. Single-stranded DNA loops are intermediates of this unwinding process. Here we show that SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding, by binding to these ssDNA intermediates. Prior to interaction with the recombination hot spot chi, RecBCD enzyme has both 3'-->5' exonuclease and a weaker 5'-->3' exonuclease activity. We show that degradation of the 5'-terminal strand at the entry site is much more extensive in the absence of SSB protein. After interaction with chi, the level of 5'-->3' exonuclease activity is increased; as expected, degradation of the 5'-strand is also elevated in the absence of SSB protein. Furthermore, we show that, in the absence of SSB protein, the RecBCD enzyme is inhibited by the ssDNA products of unwinding; SSB protein alleviates this inhibition. These results provide insight into the organization of helicase and nuclease domains within the RecBCD enzyme, and also suggest a new level at which the nuclease activity of RecBCD enzyme is controlled. Hence, they offer new insight into the role of SSB protein in the initiation phase of recombination.
Anderson
D G
DG
Genetics Graduate Group, Sections of Microbiology and Molecular and Cellular Biology, University of California Davis, Davis, CA 95616-8665, USA.
Kowalczykowski
S C
SC
D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases