A novel mode of nuclease action is revealed by the bacterial Mre11/Rad50 complex

Abstract

The Mre11/Rad50 complex is a central player in various genome maintenance pathways. Here, we report a novel mode of nuclease action found for the Escherichia coli Mre11/Rad50 complex, SbcC2/D2 complex (SbcCD). SbcCD cuts off the top of a cruciform DNA by making incisions on both strands and continues cleaving the dsDNA stem at ∼10-bp intervals. Using linear-shaped DNA substrates, we observed that SbcCD cleaved dsDNA using this activity when the substrate was 110 bp long, but that on shorter substrates the cutting pattern was changed to that predicted for the activity of a 3′-5′ exonuclease. Our results suggest that SbcCD processes hairpin and linear dsDNA ends with this novel DNA end-dependent binary endonuclease activity in response to substrate length rather than using previously reported activities. We propose a model for this mode of nuclease action, which provides new insight into SbcCD activity at a dsDNA end.

Nbs1 Converts the Human Mre11/Rad50 Nuclease Complex into an Endo/Exonuclease Machine Specific for Protein-DNA Adducts

“…The MR ortholog in E. coli SbcC/D was also shown to make double-strand breaks adjacent to 5 0 protein adducts or hairpin ends (Connelly et al, 2003;Lim et al, 2015). Similarly, we also observe dsDNA cuts on protein-DNA conjugates generated by hMRN in the presence or absence of pre-existing nicks, and the efficiency of this process depends on the length of the DNA substrate.…”

“…Similarly, we also observe dsDNA cuts on protein-DNA conjugates generated by hMRN in the presence or absence of pre-existing nicks, and the efficiency of this process depends on the length of the DNA substrate. The observation that hairpin-ended DNA stimulates SbcC/D to generate endonucleolytic double-strand breaks (Lim et al, 2015) suggests that this property of MR complex may occur at any occluded DSB end. The ability to utilize pre-existing nicks may reflect the fact that many protein-DNA adducts such as topoisomerase conjugates are usually encountered during replication, during which there are abundant nicks and gaps already present in chromosomal DNA in the nascent strands, so the physiological context of protein adducts may already involve discontinuities in the DNA.…”

“…With the label on the 5 0 blocked end, as it is located in the substrate in Figure 2A, the endonucleolytic product is not as evident compared to the 3 0 -labeled substrate in Figure 1A. One reason for the disparity in behavior on 50 versus 197 bp DNA can be the influence of DNA length on the endonucleolytic activity, as observed in the case of E. coli SbcC/D (Lim et al, 2015). To test this hypothesis with hMRN, we created 5 0 biotinylated and end-labeled 51, 101, and 197 bp DNA using PCR and carried out the endonuclease assay.…”

RecBCD, SbcCD and ExoI process a substrate created by convergent replisomes to complete DNA replication

“…Consistent with this, a recent study from Dimude et al found that SbcCD alone prevented much of the degradation that occurs in recBC mutants (Dimude, Midgley‐Smith et al , ), an observation we have confirmed in our lab. In vitro, SbcCD has been demonstrated to cleave a palindrome‐like substrate similar to that predicted to occur when replication forks bypass each other (Lim et al , ; Saathoff et al , ). Alternatively, ExoI may act independently of SbcCD to suppress the aberrant recombinational pathway.…”

Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge

“…Preparation of E. coli SbcCD. SbcCD proteins used in this study were purified and analyzed as described elsewhere 49 . Briefly, SbcCD protein complex was overexpressed from SbcCD-overexpressing plasmid pDL761 in E. coli DL776.…”