Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN

Abstract

Classical non-homologous end joining 1 (cNHEJ) and homologous recombination 2 (HR) compete for the repair of double stranded breaks of DNA during the cell cycle. HR is inhibited in G1 phase of the cell cycle, but both pathways are active in S and G2 phases. Why cNHEJ does not always outcompete HR in S and G2 phases has been unclear. Here we show that CYREN is a cell cycle specific inhibitor of cNHEJ. CYREN suppression allows cNHEJ at telomeres and intrachromosomal breaks during S and G2 phases, while cells lacking CYREN accumulate chromosomal aberrations upon damage induction, specifically outside G1 phase. CYREN acts by binding to the Ku70/80 heterodimer and preferentially inhibits cNHEJ at breaks with overhangs by protecting them. We therefore propose that CYREN is a direct cell cycle inhibitor of cNHEJ, thereby promoting error free repair by HR in cell cycle phases where sister chromatids are present.

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

“…Genetic studies in mice complement these biochemical findings as it was found that an XLF DNA-PKcs double knockout is synthetically lethal (19). Interestingly, a Ku70 knockout rescues this synthetic lethality (105).…”

“…1). A small protein called CYREN [cell cycle regulator of NHEJ (69 aa); also called MRI-2, a sub-peptide of C7orf49 (157 aa)] has been proposed to affect Ku DNA binding (not specified how) and thus favor the HR pathway choice in S/G2 (19), though the data on CYREN effects on Ku binding are conflicting (20). Signaling factors appear to be important in controlling resection, as there is evidence that the DNA damage response protein p53-binding protein 1 (53BP1) is antagonistic to end resection, acting through a number of effector proteins (21,22).…”

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

“…Ku-APLF interaction was shown to facilitate recruitment of the APLF-partner XRCC4 at damaged sites 9 and was proposed to stabilize the assembly of NHEJ factors around the DSB 19 . Notably, an A-KBM-like domain is present at the N-terminus of a recently identified inhibitor of the NHEJ pathway, CYREN(MRI), that also interacts with Ku80 20 (Figure 1a). Ku also associates with the Werner syndrome protein (WRN) that is involved in many aspects of DNA metabolism including NHEJ 21 .…”

Efficient gene correction of an aberrant splice site in β‐thalassaemia iPSCs by CRISPR/Cas9 and single‐strand oligodeoxynucleotides

“…For this aspect, we can solve the problem by utilizing a vector which can carry multiple gRNAs in the further study. Moreover, there are many other ways to improve the efficiency of gene repair, for example, adding small molecules, synchronizing the cell cycle, and adjusting delivery timing and methods . Besides, using ribonucleoprotein (RNP) delivery of Cas9 with gRNAs consistently can increase activity in cells and then enhance the efficiency …”

“…Moreover, there are many other ways to improve the efficiency of gene repair, for example, adding small molecules, synchronizing the cell cycle, and adjusting delivery timing and methods. [29][30][31] Besides, using ribonucleoprotein (RNP) delivery of Cas9 with gRNAs consistently can increase activity in cells and then enhance the efficiency. 19,31 Since gRNA can target similar sequences in a genome, it is possible to have off-targets when we use SpCas9 system.…”