The details of DNA end resection

DNA end resection is required for all recombination processes. The resection of the 5’-terminated DNA strand is required for all recombination pathways, including the SSA, synthesis-dependent strand annealing, and canonical double-strand break repair pathways. DNA end resection prevents mutagenic nonhomologous end joining. Microhomology-mediated end-joining was omitted from the scheme and text for simplicity.

Cells are exposed continuously to challenges, such as ionizing radiation and collapsed replication forks, that cause double-strand DNA breaks. Such breaks can lead to cell death or provoke chromosomal rearrangements that make a cell susceptible to cancer. As a result, cells have adapted a couple of highly efficient repair systems to keep these double-strand DNA breaks in check.

The most common repair mechanism is nonhomologous end joining, which reattaches broken DNA strands with minimal processing and without regard to missing nucleotides. The other repair mechanism, which is less error-prone, is homologous recombination, in which a single-strand overhang invades a similar or identical strand from a sister chromatid and uses it as a template to repair breaks. This repair process is the focus of a recent minireview published in the Journal of Biological Chemistry.

“The repair of DNA double-strand breaks by homologous recombination commences by nucleolytic degradation of the 5’-terminated strand of the DNA break,” which results in 3’-overhangs, explains author Petr Cejka at the University of Zurich.

In the yeast Saccharomyces cerevisiae, end resection has two steps

The first step depends on a complex of three proteins — a nuclease, Mre11; an ATPase, Rad50; and an associated protein, Xrs2 — together termed the MRX complex. Mre11 has both an endonuclease and 3’→5’ exonuclease activity.

Based on various biochemical and genetic studies, the author is in support of a short-range bidirectional resection model. He writes: “(U)pon the initial endonuclease cleavage, the Mre11 exonuclease proceeds back towards the DNA end via its 3’→5’ exonuclease activity.” This would explain how MRX is able to resect DNA with secondary structures or proteins dangling on their sides and obstructing exonucleases. “The endonuclease cut can create an entry point for long-range resection enzymes,” Cejka writes.

The second step is carried out by either the helicase/nuclease activity of Sgs1–Dna2 enzymes or the nuclease activity of Exo1 enzyme, which advances the process by resecting long stretches of DNA. The Sgs1–Dna2 tag team unwinds dsDNA in the 3’→ 5’ direction using Sgs1 helicase, while Dna2 nuclease loads onto the other strand in the 5’→3’ direction and resects ssDNA. Exo1, however, does not have to pair up with a helicase. It can degrade directly the 5’-terminated end within dsDNA.

The author also highlights the regulation of the resection process by phosphorylation of the Sae2 protein, which in turn activates Mre11. This control mechanism is carried out by cell-cycle protein kinase CDK (Cdc28) to ensure DNA is resected only in the S/G2 phase of the cycle where homologous template is available and also by DNA-damage checkpoint proteins in response to breaks. This is how cells decide if DNA resection would be a viable option, and then the “nucleases team up with the right partners to initiate (homologous recombination),” writes Cejka.

Aurelia SyngkonAurelia Syngkon is a biotechnologist and a former postdoctoral research fellow in the biochemistry and pharmacology department at New York University.