Olivia Novac

The Human Cruciform-binding Protein, CBP, Is Involved in DNA Replication and Associates in Vivo with Mammalian Replication Origins

We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 andors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3β, -ε, -γ, and -ζ isoforms with these origins was found to be ∼9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3β, -ε, -γ, and -ζ isoforms with ors8 and ors12 was found at the G1/S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G1/S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3β, -ε, -γ, and -ζ antibodies in thein vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3ε, -γ, and -ζ antibodies alone or in combination inhibited p186 replication by ∼50–80%, while anti-14-3-3β antibodies had a lesser effect (∼25–50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.

Proliferating Human Cells Hypomorphic for Origin Recognition Complex 2 and Pre-replicative Complex Formation Have a Defect in p53 Activation and Cdk2 Kinase Activation

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Δ/–) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.

14-3-3σ is a cruciform DNA binding protein and associates in vivo with origins of DNA replication

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure‐specific manner and be a member of the 14‐3‐3 protein family. CBP had been found to contain the 14‐3‐3 isoforms β, γ, ε, and ζ. Here, we show by Western blot analysis that the CBP‐cruciform DNA complex eluted from band‐shift polyacrylamide gels also contains the 14‐3‐3σ isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14‐3‐3σ isoform was able to interfere with the formation of the CBP‐cruciform DNA complex. The effect of the same anti‐14‐3‐3σ antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre‐incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non‐specific antibodies had no effect. 14‐3‐3σ was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle‐dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross‐linking, followed by immunoprecipitation with anti‐14‐3‐3σ antibody and quantitative PCR. The association of 14‐3‐3σ with the replication origins was maximal at the G1/S phase. The results indicate that 14‐3‐3σ is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding. J. Cell. Biochem. 87: 194–207, 2002.

Differential DNA binding of Ku antigen determines its involvement in DNA replication

Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins.

ABSTRACT The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G1/S border and throughout the S phase and was negligible in both G0 and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase α. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an ∼600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

Analysis of the DNA replication competence of the xrs-5 mutant cells defective in Ku86.

The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein, previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2, PCNA, DNA polymerase ϵ and δ, Primase and Topoisomerase IIα) were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oriβ) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oriβ origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 cells.