The ATR-Activation Domain of TopBP1 Is Required for the Suppression of Origin Firing during the S Phase.

The mammalian DNA replication program is controlled at two phases, the licensing of potential origins of DNA replication in early gap 1 (G1), and the selective firing of a subset of licenced origins in the synthesis (S) phase. Upon entry into the S phase, serine/threonine-protein kinase ATR (ATR) is required for successful completion of the DNA replication program by limiting unnecessary dormant origin activation. Equally important is its activator, DNA topoisomerase 2-binding protein 1 (TopBP1), which is also required for the initiation of DNA replication after a rise in S-phase kinase levels. However, it is unknown how the ATR activation domain of TopBP1 affects DNA replication dynamics. Using human cells conditionally expressing a TopBP1 mutant deficient for ATR activation, we show that functional TopBP1 is required in suppressing local dormant origin activation. Our results demonstrate a regulatory role for TopBP1 in the local balancing of replication fork firing within the S phase.

High levels of TopBP1 induce ATR-dependent shut-down of rRNA transcription and nucleolar segregation.

Nucleoli are not only organelles that produce ribosomal subunits. They are also overarching sensors of different stress conditions and they control specific nucleolar stress pathways leading to stabilization of p53. During DNA replication, ATR and its activator TopBP1 initiate DNA damage response upon DNA damage and replication stress. We found that a basal level of TopBP1 protein associates with ribosomal DNA repeat. When upregulated, TopBP1 concentrates at the ribosomal chromatin and initiates segregation of nucleolar components--the hallmark of nucleolar stress response. TopBP1-induced nucleolar segregation is coupled to shut-down of ribosomal RNA transcription in an ATR-dependent manner. Nucleolar segregation induced by TopBP1 leads to a moderate elevation of p53 protein levels and to localization of activated p53 to nucleolar caps containing TopBP1, UBF and RNA polymerase I. Our findings demonstrate that TopBP1 and ATR are able to inhibit the synthesis of rRNA and to activate nucleolar stress pathway; yet the p53-mediated cell cycle arrest is thwarted in cells expressing high levels of TopBP1. We suggest that inhibition of rRNA transcription by different stress regulators is a general mechanism for cells to initiate nucleolar stress pathway.

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding.

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.

The human Tim-Tipin complex interacts directly with DNA polymerase epsilon and stimulates its synthetic activity.

The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ε. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ε directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ε bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork.

Segregation of replicative DNA polymerases during S phase: DNA polymerase ε, but not DNA polymerases α/δ, are associated with lamins throughout S phase in human cells.

DNA polymerases (Pol) α, δ, and ε replicate the bulk of chromosomal DNA in eukaryotic cells, Pol ε being the main leading strand and Pol δ the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G(1)/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol α, δ, and ε were released from these origins upon firing. All three DNA polymerases, Mcm3 and Cdc45, but not Orc2, still formed complexes in late S phase. Reciprocal ChIP of the three DNA polymerases revealed that at G(1)/S arrest and early in S phase, Pol α, δ, and ε were associated with the same nucleoprotein complexes, whereas in late S phase Pol ε and Pol α/δ were largely associated with distinct complexes. At G(1)/S arrest, the replicative DNA polymerases were associated with lamins, but in late S phase only Pol ε, not Pol α/δ, remained associated with lamins. Consistently, Pol ε, but not Pol δ, was found in nuclear matrix fraction throughout the cell cycle. Therefore, Pol ε and Pol α/δ seem to pursue their functions at least in part independently in late S phase, either by physical uncoupling of lagging strand maturation from the fork progression, or by recruitment of Pol δ, but not Pol ε, to post-replicative processes such as translesion synthesis or post-replicative repair.

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta.

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and ß are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, ß, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and ß, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol ß inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and ß are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and ß.

Effect of 8-oxoguanine and abasic site DNA lesions on in vitro elongation by human DNA polymerase in the presence of replication protein A and proliferating-cell nuclear antigen.

DNA pol (polymerase) is thought to be the leading strand replicase in eukaryotes. In the present paper, we show that human DNA pol can efficiently bypass an 8-oxo-G (7,8-dihydro-8-oxoguanine) lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication, DNA pols associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA (proliferating-cell nuclear antigen) and of the human single-stranded DNA-binding protein RPA (replication protein A) in the modulation of the DNA synthesis and translesion capacity of DNA pol . RPA inhibited the elongation by human DNA pol on templates annealed to short primers. PCNA did not influence the elongation by DNA pol and had no effect on inhibition of elongation caused by RPA. RPA inhibition was considerably reduced when the length of the primers was increased. On templates bearing the 8-oxo-G lesion, this inhibitory effect was more pronounced on DNA replication beyond the lesion, suggesting that RPA may prevent extension by DNA pol after incorporation opposite an 8-oxo-G. Neither PCNA nor RPA had any effect on the inability of DNA pol to replicate past the AP site, independent of the primer length.

Function of TopBP1 in genome stability.

Human DNA topoisomerase IIbeta-binding protein 1 (TopBP1) and its orthologues in other organisms are proteins consisting of multiple BRCT modules that have acquired several functions during evolution. These proteins execute their tasks by interacting with a great variety of proteins involved in nuclear processes. TopBP1 is an essential protein that has numerous roles in the maintenance of the genomic integrity. In particular, it is required for the activation of ATM and Rad3-related (ATR), a vital regulator of DNA replication and replication stress response. The orthologues from yeast to human are involved in DNA replication and DNA damage response, while only proteins from higher eukaryotes are also involved in complex regulation of transcription, which is related to cell proliferation, damage response and apoptosis. We review here the recent progress in research aimed at elucidating the multiple cellular functions of TopBP1, focusing on metazoan systems.

The solution structure of the amino-terminal domain of human DNA polymerase epsilon subunit B is homologous to C-domains of AAA+ proteins.

DNA polymerases alpha, delta and epsilon are large multisubunit complexes that replicate the bulk of the DNA in the eukaryotic cell. In addition to the homologous catalytic subunits, these enzymes possess structurally related B subunits, characterized by a carboxyterminal calcineurin-like and an aminoproximal oligonucleotide/oligosaccharide binding-fold domain. The B subunits also share homology with the exonuclease subunit of archaeal DNA polymerases D. Here, we describe a novel domain specific to the N-terminus of the B subunit of eukaryotic DNA polymerases epsilon. The N-terminal domain of human DNA polymerases epsilon (Dpoe2NT) expressed in Escherichia coli was characterized. Circular dichroism studies demonstrated that Dpoe2NT forms a stable, predominantly alpha-helical structure. The solution structure of Dpoe2NT revealed a domain that consists of a left-handed superhelical bundle. Four helices are arranged in two hairpins and the connecting loops contain short beta-strand segments that form a short parallel sheet. DALI searches demonstrated a striking structural similarity of the Dpoe2NT with the alpha-helical subdomains of ATPase associated with various cellular activity (AAA+) proteins (the C-domain). Like C-domains, Dpoe2NT is rich in charged amino acids. The biased distribution of the charged residues is reflected by a polarization and a considerable dipole moment across the Dpoe2NT. Dpoe2NT represents the first C-domain fold not associated with an AAA+ protein.

Genomic changes of the 55 kDa subunit of DNA polymerase epsilon in human breast cancer.

BACKGROUND: DNA polymerases (Pols) represent potential candidates for cancer genes because of their central functions in DNA metabolism. Defects of some DNA Pols have shown cancer associations, but data on DNA polymerase (Pol) epsilon is limited. MATERIALS AND METHODS: Twenty-four human breast cancer DNA samples and four control DNA samples were examined for possible mutation in the entire coding region of the 55 kDa small subunit of the human DNA Pol epsilon gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the DNA and sequence analysis. In addition, 20 control DNAs were studied with PCR-SSCP for the end of intron 18 and exon 19 region. RESULTS: An AATT deletion was found at one location in intron 18 in 2 out of the 24 breast cancer cases (8%), but in none of the control cases. In addition, a single base transition was found in the cancer DNAs in intron 14, but the same changes were also found in the control DNAs, suggesting polymorphism. CONCLUSION: Specific changes might occur in the 55 kDa small subunit DNA sequence of DNA Pol epsilon in breast cancer. The deletion at the region of intron-exon junction may not affect the protein code, but could potentially influence splicing efficiency and expression levels, possibly impairing the function of Pol epsilon DNA.

TopBP1 and ATR colocalization at meiotic chromosomes: role of TopBP1/Cut5 in the meiotic recombination checkpoint.

Mammalian TopBP1 is a BRCT domain-containing protein whose function in mitotic cells is linked to replication and DNA damage checkpoint. Here, we study its possible role during meiosis in mice. TopBP1 foci are abundant during early prophase I and localize mainly to histone gamma-H2AX-positive domains, where DNA double-strand breaks (required to initiate recombination) occur. Strikingly, TopBP1 showed a pattern almost identical to that of ATR, a PI3K-like kinase involved in mitotic DNA damage checkpoint. In the synapsis-defective Fkbp6(-/-) mouse, TopBP1 heavily stains unsynapsed regions of chromosomes. We also tested whether Schizosaccharomyces pombe Cut5 (the TopBP1 homologue) plays a role in the meiotic recombination checkpoint, like spRad3, the ATR homologue. Indeed, we found that a cut5 mutation suppresses the checkpoint-dependent meiotic delay of a meiotic recombination defective mutant, indicating a direct role of the Cut5 protein in the meiotic checkpoint. Our findings suggest that ATR and TopBP1 monitor meiotic recombination and are required for activation of the meiotic recombination checkpoint.

Distinctive activities of DNA polymerases during human DNA replication.

The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol alpha and proliferating cell nuclear antigen displayed partial colocalization with pol delta and epsilon, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols delta and epsilon pursue their functions at least partly independently during DNA replication.

DNA polymerase epsilon associates with the elongating form of RNA polymerase II and nascent transcripts.

DNA polymerase epsilon co-operates with polymerases alpha and delta in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase epsilon and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase epsilon was also shown to UV crosslink specifically alpha-amanitin-sensitive transcripts, unlike DNA polymerase alpha that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase epsilon, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase epsilon in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase epsilon complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.

Increasing oxidative damage and loss of mismatch repair enzymes during breast carcinogenesis.

This study examined the expression of oxidative damage markers 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) and nitrotyrosine using immunohistochemical techniques. In addition, DNA topoisomerase II binding protein 1 (TopBP1) and mismatch repair proteins 2 and 6 (MSH2 and MSH6) were immunostained in a series of 80 stage I invasive breast tumours, 26 in situ breast carcinomas and 12 benign breast hyperplasias. 8-OHdG, HNE and nitrotyrosine expression were considerably weaker in hyperplasias than in in situ lesions, which, in turn, showed less oxidative damage than T1N0 tumours. Hyperplasias and in situ tumours were all, at least moderately, positive for MSH2, and nearly all were positive for MSH6. Nitrotyrosine expression was associated with HNE (P<0.0005) and 8-OHdG (P=0.041) in the T1N0 cohort. To conclude, there is increasing oxidative stress during the early steps of breast carcinogenesis. On the other hand, a significant reduction in expression of mismatch repair proteins occurs during the progression of in situ lesions to invasive tumours.

Identification of a common polymorphism in the TopBP1 gene associated with hereditary susceptibility to breast and ovarian cancer.

Besides BRCA1 and BRCA2 other genes are also likely to be involved in hereditary predisposition to breast and/or ovarian cancer. TopBP1 (topoisomerase IIbeta binding protein 1) displays sequence homology as well as functional similarities with BRCA1, and the two proteins have been suggested to function partly in the same cellular processes. TopBP1 is crucial for DNA damage and replication checkpoint controls. Based on its biological significance, we reasoned that TopBP1 is a plausible susceptibility gene for hereditary breast and/or ovarian cancer and therefore screened affected index cases from 125 Finnish cancer families for germline changes by conformation sensitive gel electrophoresis (CSGE). Altogether 19 different sequence alterations were detected. A novel heterozygous Arg309Cys variant was observed at elevated frequency in the familial cancer cases compared to healthy controls (15.2% versus 7.0%; P=0.002). Current results suggest that Arg309Cys is a commonly occurring germline alteration possibly associated with a slightly increased breast and/or ovarian cancer risk. This is the first study reporting mutation screening of the TopBP1 gene in a familial cancer material.

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D.

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3'-5' proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn2+-dependent 3'-5' exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.

Human DNA polymerase α interacts with mismatch repair proteins MSH2 and MSH6.

High fidelity of genome duplication is ensured by cooperation of polymerase proofreading and mismatch repair (MMR) activities. Here, we show that human mismatch recognizing proteins MutS homolog 2 (MSH2) and MSH6 copurify and interact with replicative Pol α. This enzyme also is the replicative primase and replicates DNA with poor fidelity. We show that MSH2 associates with known human replication origins with different dynamics than DNA polymerase (Pol α). Furthermore, we explored the potential functional role of Pol α in the mismatch repair reaction using an in vitro mismatch repair assay and observed that Pol α promotes mismatch repair. Taken together, we show that human Pol α interacts with MSH2-MSH6 complex and propose that this interaction occurs during the mismatch repair reaction.

DNA polymerase epsilon - more than a polymerase.

This paper presents a comprehensive review of the structure and function of DNA polymerase epsilon. Together with DNA polymerases alpha and delta, this enzyme replicates the nuclear DNA in the eukaryotic cell. During this process, DNA polymerase alpha lays down RNA-DNA primers that are utilized by DNA polymerases delta and epsilon for the bulk DNA synthesis. Attempts have been made to assign these two enzymes specifically to the synthesis of the leading and the lagging strand. Alternatively, the two DNA polymerases may be needed to replicate distinct regions depending on chromatin structure. Surprisingly, the essential function of DNA polymerase epsilon does not depend on its catalytic activity, but resides in the nonenzymatic carboxy-terminal domain. This domain not only mediates the interaction of the catalytic subunit with the three smaller regulatory subunits, but also links the replication machinery to the S phase checkpoint. In addition to its role in DNA replication, DNA polymerase epsilon fulfils roles in the DNA synthesis step of nucleotide excision and base excision repair, and has been implicated in recombinational processes in the cell.

Gap-directed translesion DNA synthesis of an abasic site on circular DNA templates by a human replication complex.

DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and ß are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, ß and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and ß, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, ß and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged "minicircle" DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and ß in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.

Efficient long DNA gap-filling in a mammalian cell-free system: a potential new in vitro DNA replication assay.

In vitro assay of mammalian DNA replication has been variously approached. Using gapped circular duplex substrates containing a 500-base single-stranded DNA region, we have constructed a mammalian cell-free system in which physiological DNA replication may be reproduced. Reaction of the gapped plasmid substrate with crude extracts of human HeLaS3 cells induces efficient DNA synthesis in vitro. The induced synthesis was strongly inhibited by aphidicolin and completely depended on dNTP added to the system. In cell extracts in which PCNA was depleted step-wise by immunoprecipitation, DNA synthesis was accordingly reduced. These data suggest that replicative DNA polymerases, particularly pol delta, may chiefly function in this system. Furthermore, DNA synthesis is made quantifiable in this system, which enables us to evaluate the efficiency of DNA replication induced. Our system sensitively and quantitatively detected the reduction of the DNA replication efficiency in the DNA substrates damaged by oxidation or UV cross-linking and in the presence of a potent chain terminator, ara-CTP. The quantitative assessment of mammalian DNA replication may provide various advantages not only in basic research but also in drug development.