DNA Replication In Egg Extracts Research Articles

RECQL4 is not critical for firing of human DNA replication origins

Human RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, the Xenopus laevis homolog of RECQL4 has been implicated in initiating DNA replication in egg extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (knock-out; KO) or expression of a full-length, mutant protein lacking helicase activity (helicase-dead; HD). Interestingly, both the RECQL4 KO and HD cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed increased rates in the RECQL4 KO cells, which might be indicative of decreased origin firing efficiency. Our results are consistent with human RECQL4 having a less critical role in firing of DNA replication origins, than its budding yeast homolog Sld2.

Initiation of DNA Replication Requires the RECQL4 Protein Mutated in Rothmund-Thomson Syndrome

How the replication machinery is loaded at origins of DNA replication is poorly understood. Here, we implicate in this process the Xenopus laevis homolog (xRTS) of the RECQL4 helicase mutated in Rothmund-Thomson syndrome. xRTS, which bears homology to the yeast replication factors Sld2/DRC1, is essential for DNA replication in egg extracts. xRTS can be replaced in extracts by its human homolog, while RECQL4 depletion from mammalian cells induces proliferation failure, suggesting an evolutionarily conserved function. xRTS accumulates on chromatin during replication initiation, after prereplication-complex (pre-RC) proteins, Cut5, Sld5, or Cdc45 but before replicative polymerases. xRTS depletion suppresses the loading of RPA, the ssDNA binding protein that marks unwound origins before polymerase recruitment. However, xRTS is unaffected by xRPA depletion. Thus, xRTS functions after pre-RC formation to promote loading of replication factors at origins, a previously unrecognized activity necessary for initiation. This role connects defective replication initiation to a chromosome-fragility disorder.

Mcm2 Is a Direct Substrate of ATM and ATR during DNA Damage and DNA Replication Checkpoint Responses

In vertebrates, ATM and ATR are critical regulators of checkpoint responses to damaged and incompletely replicated DNA. These checkpoint responses involve the activation of signaling pathways that inhibit the replication of chromosomes with DNA lesions. In this study, we describe the isolation of a cDNA encoding a full-length version of Xenopus ATM. Using antibodies against the regulatory domain of ATM, we have identified the essential replication protein Mcm2 as an ATM-binding protein in Xenopus egg extracts. Xenopus Mcm2 underwent phosphorylation at Ser(92) in response to the presence of double-stranded DNA breaks or DNA replication blocks in egg extracts. This phosphorylation involved both ATM and ATR, but the relative contribution of each kinase depended upon the checkpoint-inducing DNA signal. Furthermore, both ATM and ATR phosphorylated Mcm2 directly at Ser(92) in cell-free kinase assays. Immunodepletion of both ATM and ATR abrogated the checkpoint response that blocks chromosomal DNA replication in egg extracts containing double-stranded DNA breaks. These experiments indicate that ATM and ATR phosphorylate the functionally critical replication protein Mcm2 during both DNA damage and replication checkpoint responses in Xenopus egg extracts.

Xenopus Drf1, a Regulator of Cdc7, Displays Checkpoint-dependent Accumulation on Chromatin during an S-phase Arrest

We have cloned a Xenopus Dbf4-related factor named Drf1 and characterized this protein by using Xenopus egg extracts. Drf1 forms an active complex with the kinase Cdc7. However, most of the Cdc7 in egg extracts is not associated with Drf1, which raises the possibility that some or all of the remaining Cdc7 is bound to another Dbf4-related protein. Immunodepletion of Drf1 does not prevent DNA replication in egg extracts. Consistent with this observation, Cdc45 can still associate with chromatin in Drf1-depleted extracts, albeit at significantly reduced levels. Nonetheless, Drf1 displays highly regulated binding to replicating chromatin. Treatment of egg extracts with aphidicolin results in a substantial accumulation of Drf1 on chromatin. This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin. These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin. Abrogation of this pathway also leads to a large increase in the binding of Cdc45 to chromatin. This increase is substantially reduced in the absence of Drf1, which suggests that regulation of Drf1 might be involved in the suppression of Cdc45 loading during replication arrest. We also provide evidence that elimination of this checkpoint causes resumed initiation of DNA replication in both Xenopus tissue culture cells and egg extracts. Taken together, these observations argue that Drf1 is regulated by an intra-S-phase checkpoint mechanism that down-regulates the loading of Cdc45 onto chromatin containing DNA replication blocks.

Identification and characterization of a Xenopus homolog of Dbf4, a regulatory subunit of the Cdc7 protein kinase required for the initiation of DNA replication.

Dbf4 is a regulatory subunit for the Cdc7 protein kinase that is required for the initiation of eukaryotic DNA replication, but the precise roles of Dbf4-Cdc7 remain to be determined. Here we identified a Xenopus homolog of Dbf4 (XDbf4) and characterized XDbf4 and Xenopus Cdc7 (XCdc7) in Xenopus egg extracts. XDbf4 formed a complex with XCdc7 in egg extracts and activated XCdc7 kinase activity in vitro. In contrast with Dbf4 in yeast and mammalian cultured cells, the XDbf4 levels in egg extracts did not change during the cell cycle progression. XDbf4 was a phosphoprotein in interphase extracts, and was apparently hyperphosphorylated in cytostatic factor (CSF)-mediated, metaphase-arrested extracts and in mitotic extracts. However, the hyperphosphorylation of XDbf4 did not seem to affect the level of kinase activation, or chromatin binding of the XDbf4-XCdc7 complex. Upon release from CSF-arrest, XDbf4 was partially dephosphorylated and bound to chromatin. Interestingly, XDbf4 was loaded onto chromatin before XCdc7 during DNA replication in egg extracts. These results suggest that the function of XDbf4-XCdc7 during the early embryonic cell cycle is regulated in a manner distinct from that during the somatic cell cycle.