Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation

Abstract

DNA recombination pathways are cell cycle regulated to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5'-strand resection at DNA double strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11–Rad50–Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase/topoisomerase complex Sgs1–Top3–Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting crosstalk between Dna2- and Exo1-dependent resection pathways.

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

“…When the fork demise occurs, however, broken forks could be repaired by DSB resection by homologous recombination (53). In this case, long range resection by Dna2 is affected as recently demonstrated (39), which ultimately leads to a reduced level of ssDNA. In both cases, the amount of ssDNA could be reduced to a level that fails to elicit the S-phase checkpoint activation.…”

“…Based on our biochemical results, we hypothesized that the absence of the N-terminal 45-kDa domain could lead to defects in either Okazaki fragment processing or aberrant resection of DSBs (39). It is reasonable to assume that the two events described above are inseparable as defective Okazaki fragment processing could result in increased DSB formation (36).…”

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

“…In Schizosaccharomyces pombe, periodic expression of the CtIP homolog Ctp1 is also relevant for the cell cycle-dependent regulation of resection (16), and this mechanism seems to be conserved in mammalian cells (17). A second CDK target is the Dna2 nuclease, whose phosphorylation stimu-lates its recruitment to DSBs (18). The above findings suggested a straightforward model in which a phospho-dependent switch at the G 1 -S transition turns on resection.…”

Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation

“…According to Gene Ontology annotations of genes that were expressed significantly higher in the genomic-unstable sample clone 6 P3 compared with clone 2 P3 and clone 8 P3, 14.7% genes enriched in Gene Ontology annotations were associated with cell cycle, 13.5% were associated with cancer and 10.9% were associated with cellular motility. These data were consistent with the pathway and network analyses using the same gene data sets (data not shown), indicating that clone 6 P3 was much more active 0 0 1 42 42 2 24 1246 52 1 83 83 23 1163 51 3 177 13653 77 71 3010 42 106 10642 100 4 7 721 103 1 19 19 6 702 117 5 17 444 26 0 0 0 17 444 26 6 56 3030 54 6 212 35 50 2819 56 7 2 41 21 2 41 Figure 7), including CCND1, NFKB2, IL1B, IL6, CDK1, and PTGS2, are mainly linked to the biological functions of cell cycle and immune response, [24][25][26][27][28] and may serve as biomarkers for the identification of hUCMSCs with the genomic instability potential at early passages. These molecules may also be interesting because of possible clinical applications.…”