The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA

Abstract

Hairpin structures can inhibit DNA replication and are intermediates in certain recombination reactions. We have shown that the purified SbcCD protein of Escherichia coli cleaves a DNA hairpin. This cleavage does not require the presence of a free (3' or 5') DNA end and generates products with 3'-hydroxyl and 5'-phosphate termini. Electron microscopy of SbcCD has revealed the "head-rod-tail" structure predicted for the SMC (structural maintenance of chromosomes) family of proteins, of which SbcC is a member. This work provides evidence consistent with the proposal that SbcCD cleaves hairpin structures that halt the progress of the replication fork, allowing homologous recombination to restore DNA replication.

Figure 1

Trapping the initial hairpin cleavage products generated by SbcCD. (A) An autoradiograph illustrating the DNA fragments produced by SbcCD over a 30-min time period. SbcCD was incubated with 3′-end-labeled hairpin substrate (3′HP78), in the presence of ATP at 37°C, for 0 min (lane b), 1 min (lane c), 5 min (lane d), 10 min (lane e), or 30 min (lane f). 3′HP78 also was incubated for 30 min in the absence of SbcCD (lane a). Early products produced by SbcCD are indicated. (B) Effect of various nucleotides on the SbcCD hairpin-nuclease activity. SbcCD was incubated with 3′-end-labeled hairpin substrate (3′HP78), at 16°C for 30 min, in the presence of various nucleotides. Reactions were performed in the presence of ATP (lane b), dATP (lane c), ADP (lane d), ATPγS (lane e), GTP (lane f), GDP (lane g), or no nucleotide (lane h). 3′HP78 also was incubated with ATP in the absence of SbcCD (lane a). Products were visualized by gel electrophoresis. The sizes of the fragments produced in the presence of ATPγS (lane e) and GTP (lane f) are indicated.

Figure 2

5′- and 3′-labeled hairpin substrates incubated with SbcCD in the presence of ATPγS. ∗, the position of the ³²P-label. The SbcCD product is flanked by A/G sequencing ladders. Fragments arising as a result of chemical cleavage at the three guanosines adjacent to the hairpin loop are indicated. (A) 3′-end-labeled hairpin (3′HP78) was incubated with SbcCD in the presence of ATPγS, at 16°C for 30 min, and reaction products were resolved on a polyacrylamide gel (lane b). A/G sequencing ladders of 3′HP78 are shown in lanes a and c. (B) 5′-end-labeled hairpin (5′HP78) was incubated with SbcCD in the presence of ATPγS, at 16°C for 30 min, and reaction products were resolved on a polyacrylamide gel (lane b). ∗, the position of the ³²P-label. A/G sequencing ladders of 5′HP78 are shown in lanes a and c. A one-and-one-half base allowance was made to compensate for the nucleoside eliminated in the sequencing reaction.

Figure 3

Analysis of hairpin cleavage termini. (A) 5′ termini: 3′-end-labeled hairpin (3′HP78) was digested with SbcCD for 30 min at 16°C, in the presence of ATPγS, and the reaction products then were incubated with (lane a) or without (lane b) shrimp alkaline phosphatase. SbcCD cleavage products are indicated. A 1:1 mix of the samples loaded to lanes a and b were analyzed in lane c to demonstrate that the mobility shift observed was not a lane-specific artefact. (B) 3′-termini: 5′-end-labeled hairpin (5′HP78) was digested with SbcCD, in the presence of ATPγS, and the reaction products were incubated in the absence (lane a) or presence (lane b) of terminal transferase.

Figure 4

SbcCD cleaves a covalently closed dumbbell substrate. (A) Internally labeled, covalently closed dumbbell substrate (ccDB98) was incubated without (lane a) or with (lane b) SbcCD in the presence of ATPγS, at 16°C for 30 min. ∗, the position of the ³²P-label. Reaction products then were resolved on a denaturing polyacrylamide gel. The 49- and 98-nt fragments produced are indicated (49 and ncDB98). (B) Preparative 10% polyacrylamide gel showing the single product obtained after the ligation of ncDB98. The upper band is covalently closed dumbbell (ccDB98), the lower band is nicked-circular dumbbell (ncDB98), which failed to ligate.

Figure 5

Electron micrograph of SbcCD protein. SbcCD was visualized by using the low angle rotary shadowing method (Magnification, ×150,000.) Structures can be seen that consist of two globular domains linked by a long filamentous rod.