Overexpression, Purification, and Characterization of the SbcCD Protein from Escherichia coli

Abstract

The sbcC and sbcD genes mediate palindrome inviability in Escherichia coli. The sbcCD operon has been cloned into the plasmid pTrc99A under the control of the strong trc promoter and introduced into a strain carrying a chromosomal deletion of sbcCD. The SbcC and SbcD polypeptides were overexpressed to 6% of total cell protein, and both polypeptides copurified in a four-step purification procedure. Purified SbcCD is a processive double-strand exonuclease that has an absolute requirement for Mn 2؉ and uses ATP as a preferred energy source. Gel filtration chromatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after purification and that this dissociation is reversed by the addition of Mn 2؉ . We demonstrate that SbcD has the potential to form a secondary structural motif found in a number of protein phosphatases and suggest that it is a metalloprotein that contains the catalytic center of the SbcCD exonuclease.

Accumulation of the Lipid A Precursor UDP-2,3-diacylglucosamine in an Escherichia coli Mutant Lacking the lpxH Gene

“…P. aeruginosa LpxH is 46% identical and 61% similar (E value of 10 Ϫ59 ) to E. coli LpxH, whereas P. aeruginosa LpxH2 has 28% identity and 39% similarity (E value of 0.009) to a 119-amino acid segment of E. coli LpxH. Despite these differences, all the LpxH homologs contain a phosphoesterase signature sequence common to a family of DNA repair exonucleases (MRE11/RAD32 and SbcD) (35)(36)(37), mammalian and bacterial serine/threonine protein phosphatases (38 -40), mammalian and plant purple acid phosphatases (41,42), and the E. coli periplasmic 5Ј-nucleosidase (43). This signature sequence, DXH(X) n GDXXD(X) n GNH(D/E) (where n is ϳ25 residues), provides a scaffold for an active site dinuclear metal center (38,41,(43)(44)(45)(46).…”

Structure of the Rad50·Mre11 DNA Repair Complex fromSaccharomyces cerevisiae by Electron Microscopy

“…After several trials, two factors important for maintenance of the complex were identified: solution pH and the addition of manganese (Mn 2ϩ ). Manganese is an essential cofactor for Mre11 and all of its homologs for nuclease activity and is also a crucial factor in stabilizing the SbcCD complex from E. coli (31). We hypothesized that it might play a similar role for the Rad50⅐Mre11 complex.…”

Instability of repetitive DNA sequences: The role of replication in multiple mechanisms

“…The sbcD gene encodes the nuclease component of the SbcCD complex of E. coli (62). In vitro, SbcCD possesses ssDNA endonuclease and double-strand DNA (dsDNA) exonuclease activities (63,64) and can cleave hairpin structures (65). In vivo, SbcCD appears to introduce DSBs at palindromic sequences (66,67).…”