Abstract
The Mre11–Rad50 complex is a DNA double-strand break sensor that cleaves blocked DNA ends and hairpins by an ATP-dependent endo/exonuclease activity for subsequent repair. For that, Mre11–Rad50 complexes, including the Escherichia coli homolog SbcCD, can endonucleolytically cleave one or both strands near a protein block and process free DNA ends via a 3′-5′ exonuclease, but a unified basis for these distinct activities is lacking. Here we analyzed DNA binding, ATPase and nuclease reactions on different DNA substrates. SbcCD clips terminal bases of both strands of the DNA end in the presence of ATPγS. It introduces a DNA double-strand break around 20–25 bp from a blocked end after multiple rounds of ATP hydrolysis in a reaction that correlates with local DNA meltability. Interestingly, we find that nuclease reactions on opposing strands are chemically distinct, leaving a 5′ phosphate on one strand, but a 3′ phosphate on the other strand. Collectively, our results identify an unexpected chemical variability of the nuclease, indicating that the complex is oriented at a free DNA end and facing a block with opposite polarity. This suggests a unified model for ATP-dependent endo- and exonuclease reactions at internal DNA near a block and at free DNA ends.
Highlights
The maintenance and accurate replication of genomes are fundamental processes in all kingdoms of life
DNA damage and complications in DNA replication can cause genomic alterations ranging from point mutations to gross chromosomal aberrations and aneupleuidy, which in humans is associated with the development of cancer and other diseases
To test to the role of DNA topology on ATP hydrolysis we measured the stimulatory effect of a 5.4 kb plasmid in (i) supercoiled (ii) relaxed (iii) linearized and (iv) singlestranded state
Summary
The maintenance and accurate replication of genomes are fundamental processes in all kingdoms of life. In all phylogenetic kingdoms the propagation and maintenance of the genome critically depends on various pathways that detect, signal and repair DNA damage and deal with replicative stress [1]. The nuclease Mre and the ATPase Rad form an evolutionary highly conserved complex, which is involved in genome maintenance and replication by detecting and processing DNA double-strand breaks, hairpins and other abnormal terminal DNA structures [2]. MRN/X and SbcCD detect DNA end structures and can process blocked or obstructed DNA ends and hairpins to make them accessible for DSB repair [4,5,6,7,8]. The main DSB repair pathways following DNA end processing by MRN/X are various end joining reactions and homologous recombination (HR) [9,10]
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