Chromosomal breakage results from a DNA double strand break (DSB), and is repaired to maintain and restore genetic integrity, principally through two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by nucleolytic resection of a DSB in the presence of cyclin-dependent kinase 1 (Cdk1) activity. DSB repair through HR is dependent on Rad52, and can be error-free when a sister chromatid is used as a template for repair. However, HR is mutagenic when any other template is used for repair. Loss of nucleotides adjacent to the DSB is a feature of repair through NHEJ. There is co-relation between Cdk1 activity and the presence of a sister chromatid. The research question was, in addition to Cdk1 activity is the presence of an intact sister chromatid a requirement to initiate DSB repair with the HR pathway.
Cdk1 activity peaks during mitosis in the presence of an intact sister chromatid. To study DSB resection and repair in cells arrested in either mitotic metaphase or telophase when Cdk1-Clb2 was active, conditional alleles were constructed in a eukaryotic haploid budding yeast model of HR. The model permitted simultaneous induction of a single site-specific DSB in cells that were synchronised to a phase of the cell division cycle. Physical monitoring of the kinetics of DSB formation, nucleolytic resection of adjacent DNA, and DSB repair, was achieved by probing Southern membranes after restriction enzyme digestion of extracted genomic DNA from time courses.
Sister chromatids were segregated during telophase arrest induced by either Cdc14 or Cdc15 depletion. Metaphase arrest was achieved with Cdc20 depletion, either directly, or indirectly by activation of the spindle assembly checkpoint by inhibition of microtubule polymerisation. Sister chromatids were unsegregated and physically attached through cohesin during metaphase.
The absence of an intact sister chromatid did not prevent DSB repair with the HR pathway during telophase. Nucleolytic resection was observed in the presence or absence of an intra-chromosomal homologous but non-identical DNA repair template. The DSB cut site did not become resistant to cycles of re-cleavage through loss of adjacent nucleotides. DSB repair by HR was dependent on Rad52. The kinetics of nucleolytic resection adjacent to the DSB, and repair by HR, were similar during telophase and metaphase.
This is the first study to report the observation that the availability of the sister chromatid is not a requirement to promote DSB repair with the HR pathway during telophase. Initiation of HR occurs despite segregated sister chromatids, even in the absence of a non-identical homologous DNA donor template, with inherently mutagenic repair by HR. This unexpected discovery has important clinical implications to the pathogenesis of chromosomal translocations and oncogenesis, and tumour progression with repair of treatment-induced DSBs.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.