Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination

Abstract

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.

Dbf4-Dependent Kinase Finetunes INO80 Function at Chromosome Replication Origins

“…The objective of this study was to expand our knowledge about DDK-dependent phosphorylation events and to identify phosphoproteins using a global proteomic approach, which included phosphopeptide enrichment in the presence and absence of DDK function in vivo. A comparable approach was recently published, in which DDK phosphoproteomes were identified in M-phase-arrested budding yeast cells (Galanti et al, 2024). Given that our primary interest was in chromatin dynamics and associated processes during replication, we reasoned that it would be most informative to analyse phosphoproteomes of nuclear fractions and specifically determine phosphoproteomes of samples in S phase.…”

“…In contrast, DDK’s kinase Cdc7 has only one activator, Dbf4, which accumulate in S phase, where it is essential for the initiation of replication (Bousset et al, 1998; Donaldson et al, 1998; Gillespie et al, 2022; Jiang et al, 1999; Sheu et al, 2006, 2010). In addition to DDK’s pivotal role in S phase, Dbf4 levels remain high until the metaphase-to-anaphase transition and DDK is involved in processes like meiotic recombination (Sasanuma et al, 2008; Wan et al, 2008), chromosome segregation (Argunhan et al, 2017; Challa et al, 2019; He et al, 2020; Matos et al, 2008), DNA double-strand break repair (Galanti et al, 2024; Princz et al, 2017) and the block to over-replication (Reusswig et al, 2016). Interestingly, DDK was found to interact with ORC at early-replicating origins in the G1 phase (Duncker et al, 2002; Fang et al, 2017; Pasero et al, 1999).…”