Cdc7-Dbf4 and the Human S Checkpoint Response to UVC

Abstract

The S checkpoint response to ultraviolet radiation (UVC) that inhibits replicon initiation is dependent on the ATR and Chk1 kinases. Downstream effectors of this response, however, are not well characterized. Data reported here eliminated Cdc25A degradation and inhibition of Cdk2-cyclin E as intrinsic components of the UVC-induced pathway of inhibition of replicon initiation in human cells. A sublethal dose of UVC (1 J/m 2 ), which selectively inhibits replicon initiation by 50%, failed to reduce the amount of Cdc25A protein or decrease Cdk2-cyclin E kinase activity. Cdc25A degradation was observed after irradiation with cytotoxic fluences of UVC, suggesting that severe inhibition of DNA chain elongation and activation of the replication checkpoint might be responsible for the UVC-induced degradation of Cdc25A. Another proposed effector of the S checkpoint is the Cdc7-Dbf4 complex. Dbf4 interacted weakly with Chk1 in vivo but was recognized as a substrate for Chk1-dependent phosphorylation in vitro. FLAG-Dbf4 formed complexes with endogenous Cdc7, and this interaction was stable in UVC-irradiated HeLa cells. Overexpression of FLAG-or Myc-tagged Dbf4 abrogated the S checkpoint response to UVC but not ionizing radiation. These findings implicate a Dbf4-dependent kinase as a possible target of the ATR-and Chk1-dependent S checkpoint response to UVC.Human cells continuously experience damage to DNA from both reactive cellular metabolites and environmental sources. If damaged DNA is not repaired before DNA replication, mutations and chromosomal aberrations might ensue. To maintain high fidelity replication of the genome, human cells have evolved a number of biochemical pathways that respond to perturbations in DNA structure and facilitate error-free replication of damaged DNA (1). The S checkpoint maintains genomic stability by transiently inhibiting the initiation of new replicons and thereby limiting the number of active replication forks that encounter DNA lesions. This transient inhibition of replicon initiation should provide more time for DNA repair pathways to recognize and eliminate DNA damage prior to replication.Replicon initiation is a biochemically and genetically conserved process across eukaryotic phyla that requires the stepwise formation of pre-replication complexes at origins of DNA replication (2). The six-subunit origin recognition complex binds to replication origins throughout the cell cycle and acts as a landing pad for Cdc6. Origin recognition complex and Cdc6 collaborate with the chaperone protein Cdt1 to load the sixsubunit MCM 4 complex into the developing pre-replication complexes. Mcm2-7 forms a hexameric structure, which is hypothesized to be the mammalian DNA helicase that unwinds duplex DNA to allow DNA polymerases to synthesize daughter strands. The origin recognition complex-Cdc6-Cdt-MCM prereplication complex is regulated by two S phase kinase complexes, a cyclin-dependent kinase (CDK) and a Dbf4-dependent kinase (DDK). S phase CDKs phosphorylate Cdc6, which results in its degradatio...

ATM and ATR signaling at a glance

“…Chk1 activation stabilizes the chromatin-bound CDC7-DBF4 apoptosis signalregulating kinase (ASK) complex, which assists in replication and origin firing (Yamada et al, 2013). Chk1 affects progression through S phase at the level of origin firing, replication elongation and fork integrity (Brown and Baltimore, 2003;Heffernan et al, 2007;Petermann et al, 2006Petermann et al, , 2010Segurado and Diffley, 2008;Zhao et al, 2002). In addition, activated Chk1 also has an effect on S-phase progression through phosphorylation of CDC25 and regulation of cyclin-dependent kinases (CDKs) (Sanchez et al, 1997).…”

Chk1 promotes replication fork progression by controlling replication initiation

“…Although it is currently not clear whether Chk1 regulates Cdc7 in vivo (20,21), Cdc7 activity is known to promote origin firing. Accordingly, Cdc7 depletion had a similar effect to Cdk inhibition in rescuing replication fork speeds in Chk1-deficient cells (Fig.…”

“…Firstly it inhibits the cyclin-dependent kinase Cdk2 (in complex with Cyclin A or E) by phosphorylation of its activating phosphatase Cdc25A, thus marking Cdc25A for degradation (19). Secondly Chk1 can phosphorylate and inhibit the initiation kinase Cdc7/Dbf4 (Dbf4-dependent kinase, DDK) (20), although it is currently not established that Cdc7 is down-regulated during replication stress (21). Both Cdk2 and Cdc7 phosphorylate the preRC to facilitate loading of the replicative helicase cofactor Cdc45 and thus origin activation (22).…”

Controlling DNA replication origins in response to DNA damage – inhibit globally, activate locally

“…There is evidence that CDK activity is inhibited in response to DNA damage in human cells through the CHK1-dependent degradation of Cdc25A, a phosphatase that is required for CDK activation in S phase (Falck et al, 2001). There is evidence from Xenopus egg extracts and human cells that DDK might also be regulated in response to the activation of DNA damage checkpoints during S phase (Costanzo et al, 2003;Heffernan et al, 2007;Matsuoka et al, 2007).…”