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THE CELL NUCLEUS AND DNA REPLICATION


Initiation of DNA replication: dissection of a human DNA replication origin.


Fiorenzo Peverali


The correct duplication of the genome is critical for the survival of cells and organisms. It is not surprising, therefore, that the replication process is subjected to a number of control mechanisms aimed at controlling the fidelity of the replication process and to ensure that any DNA sequence is duplicated once and only once during the cell cycle. A critical point of DNA replication is the selection of the sites where DNA synthesis starts. Studies in model systems, such as bacteria, viruses and lower eukaryotes (yeast), have bolstered the “replicon” model according to which DNA synthesis starts in correspondence of specific DNA sequences called “origins” that are recognized and bound by a specific protein, the “initiator”, that loads of the replication machinery onto DNA. Several data argue against the general validity of the model suggesting that chromatin contexts or additional elements, rather than sequence, direct the selection of DNA replication start sites. Thus, in mammalian cells DNA synthesis would start in broad, poorly defined, “initiation zones”. This probably reflects the fact that the mammalian initiator, called ORC for Origin Recognition Complex, does not show any intrinsic strong specificity of binding to DNA.

Our laboratory, in collaboration with the group of Arturo Falaschi at the ICGBE in Trieste, has significantly contributed to the analysis of DNA replication origins in human cells trying to address the contention about the specificity of initiation. We have isolated one of the few human DNA replication origin identified so far. This origin, called LamB2-ori, has been mapped to a narrow (400 bp) DNA sequence that contains the 3’-end of the lamin B2 gene and the CpG island associated to the Timm13 gene. A multiprotein complex is assembled on the origin in a cell cycle-depended and sequence specific manner. This complex (OBP) contains proteins of the pre-replicative complex (ORC, Cdc6, MCM) but also proteins that control the topological status of DNA (Topoisomerase I and II). The region covered by the OBP comprises the start sites of DNA replication - OBR. We have mapped, with nucleotide precision, the start sites of the leading strands synthesis. Thus our analysis clearly supports the notion that, at least in the case of a subset of origins including LamB2, DNA replication starts at a precise sequence rather than in an “initiation zone”.

More recently we have focused our efforts on the dissection of the origin trying to identify the critical sequence elements. We have shown that a 1,2 kb DNA fragment comprising the start site of DNA replication along with the OBP and the CpG island associated with the TIMM13 promoter behaves as an origin of DNA replication when integrated at ectopic positions of the human genome. By testing the activity of different mutants we have found that the activity of the origin depends both on sequences proximal to the OBR, within the OBP, and on the CpG island. Thus, our analysis demonstrates that specific sequence elements are required for the activity of the origin.




Figure 4. Schematic representation of the genomic region containing the origin of DNA replication associated to the Lamin B2 gene (LamB2 ori). Below it is shown the 1,2 kb fragment comprising the origin and the CpG island associated to the TIMM13 promoter.
Bottom left: analysis of an ectopic origin that is as active as the endogenous LamB2 ori. Bottom right: activity of ectopic origins integrated at random positions of the human genome. The red bar is the activity of the endogenous LamB2 ori.



In order to investigate more in detail the role of sequence elements in the origin activity we have recently developed an assay in which origin mutants are tested in the same chromosomal position. DNA replication origins are key elements in the construction of artificial chromosomes and expression vectors to be employed in gene therapy approaches. Experiments are underway in our laboratory to confirm the hypothesis that inclusion of functional replicators in gene therapy vectors may provide a tool for stabilizing the transgenes expression patterns.

This assay is based on the Cre/Lox technology and takes advantage of human cell lines, developed in our laboratory, bearing a single Lox site that acts as a recipient for the transfected origin fragment. By means of this approach we can compare the activity of different LamB2 mutants integrated in the same chromosomal context. We have found that the activity of the wild type 1,2 kb DNA fragment comprising the start site of DNA replication depends on the site of integration. Moreover, the CpG island is important for the full activity of the origin even if its relevance depends on the integration sites. Further experiments are underway to identify all the sequence elements that control the activity of the origin.


References

  1. Falaschi A, Abdurashidova G, Sandoval O, Radulescu S, Biamonti G, Riva S. (2007) Molecular and structural transactions at human DNA replication origins. Cell Cycle. 6(14):1705-12.
  2. Abdurashidova G, Radulescu S, Sandoval O, Zahariev S, Danilov M, Demidovich A, Santamaria L, BiamontiI G, Riva S, and Falaschi A. (2007) Functional interactions of DNA topoisomerases with a human replication origin.- The EMBO Journal . 26(4): 998-1009
  3. Paixao S, Colaluca IN, Cubells M, Peverali FA, Destro A, Giadrossi S, Giacca M, Falaschi A, Riva S, Biamonti G. (2004) Modular structure of the human lamin B2 replicator. Mol Cell Biol. 24:2958-67.
  4. Biamonti G, Paixao S, Montecucco A, Peverali FA, Riva S, Falaschi A. (2003) Is DNA sequence sufficient to specify DNA replication origins in metazoan cells? Chromosome Res. 11:403-12.
  5. Gabellini D, Colaluca IN, Vodermaier HC, Biamonti G, Giacca M, Falaschi A, Riva S, Peverali FA. (2003) Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression. EMBO J. 22:3715-24.
  6. de Stanchina E, Gabellini D, Norio P, Giacca M, Peverali FA, Riva S, Falaschi A, Biamonti G. (2000) Selection of homeotic proteins for binding to a human DNA replication origin. J Mol Biol. 299:667-80.
  7. Abdurashidova G, Deganuto M, Klima R, Riva S, Biamonti G, Giacca M, Falaschi A. (2000) Start sites of bidirectional DNA synthesis at the human lamin B2 origin. Science. 287:2023-6.
  8. Abdurashidova G, Riva S, Biamonti G, Giacca M, Falaschi A. (1998) Cell cycle modulation of protein-DNA interactions at a human replication origin. EMBO J. 17:2961-9.

 

 


Dynamics of replication factories

Alessandra Montecucco


In eukaryotic cells DNA replication takes place in specific sub-nuclear districts called replication factories or RFs. In addition to replicative proteins RFs contain factors that control the fidelity of the replication process and chromatin assembly. The number, size and distribution of RFs change during the S-phase according to a precise program. Five distribution patterns of RFs have been described each one specific of a different moment of the S-phase. Each pattern is associated with the replication of specific portions of the genome. Thus, for instance, euchromatin is replicated in early S-phase in a large number of small factories. Replication of pericentric heterochromatin occurs in large factories adjacent to the nucleoli or to the nuclear envelope. Finally, large heterochromatic masses at the centromeres are replicated in very large RFs.

In our laboratory we contributed to the analysis of several aspects of RFs. Initially, we identified a protein motif in the N-terminal regulatory domain of DNA ligase I that is necessary for the recruitment of the enzyme to sites of active DNA replication. This motif is shared with a number of proteins found in RFs and corresponds to a binding site for the replicative factor PCNA. On the basis of this analysis we have hypothesized a major role of PCNA in the assembly of replication factories.




Figure 5. Confocal laser microscopy analysis of the distribution of DNA ligase I (a marker of replication factories) and of sites of incorporation of BrdU (replication foci). The merged image highlights the colocalization of the two markers.

 

We have also mapped in the same N-terminal regulatory domain of DNA ligase I, close to the PCNA binding motif, a number of residues that are phosphorylated in a cell-cycle-dependent manner. The function of these sites is still matter of investigation. However, we have described a temporal order in the phosphorylation of the different residues as if these post-translational modifications could modulate the association of the enzyme with replication complexes.

We have also hypothesized that the activity and distribution of replication factories could be subjected to several control pathways and that phosphorylation is part of these regulatory circuits. To verify if this is indeed the case we have investigated the dynamic of RFs in response to treatments with anticancer drugs that elicit the DNA damage checkpoint response. We found that the anti-tumor drug etoposide, a poison of DNA topoisomerase II, induces the dispersion of replicative proteins from sites of DNA synthesis. This phenomenon requires the activity of the checkpoint pathway identified by the apical ATR kinase and the downstream Chk1 kinase. Our data are consistent with a model whereby etoposide induces double-stranded DNA breaks (DSBs) behind the replication fork. DSBs are then converted to single-stranded DNA regions covered by the single-strand DNA binding protein RPA. RPA, in turns activates the ATR checkpoint pathway which prevents firing of late origins. Since etoposide does not lead to an immediate block of DNA synthesis, already activated replicons are fully replicated even in the presence of the drug.
We have also found that specific pathways are required for inhibition of early and mid-late RFs. In fact, the effect of etoposide on early RFs requires the activity of the NBS1 protein, which is instead dispensable in the case of mid-late S-phase factories. Both early and mid-late RFs require the Chk1 function to by inhibited by etoposide.

References

  1. Montecucco A, Biamonti G. (2006) Cellular response to etoposide treatment. Cancer Lett. 252(1):9-18
  2. Rossi R, Lidonnici MR, Soza S, Biamonti G, Montecucco A. (2006) The dispersal of replication proteins after Etoposide treatment requires the cooperation of Nbs1 with the ataxia telangiectasia Rad3-related/Chk1 pathway. Cancer Res. 66:1675-83.
  3. Lidonnici MR, Rossi R, Paixao S, Mendoza-Maldonado R, Paolinelli R, Arcangeli C, Giacca M, Biamonti G, Montecucco A. (2004) Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle. J Cell Sci. 117:5221-31.
  4. Ferrari G, Rossi R, Arosio D, Vindigni A, Biamonti G, Montecucco A. (2003) Cell cycle-dependent phosphorylation of human DNA ligase I at the cyclin-dependent kinase sites. J Biol Chem. 278:37761-7.
  5. Frouin I, Montecucco A, Biamonti G, Hubscher U, Spadari S, Maga G. (2002) Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins. EMBO J. 21:2485-95.
  6. Montecucco A, Rossi R, Ferrari G, Scovassi AI, Prosperi E, Biamonti G. (2001) Etoposide induces the dispersal of DNA ligase I from replication factories. Mol Biol Cell. 12:2109-18.
  7. Rossi R, Villa A, Negri C, Scovassi I, Ciarrocchi G, Biamonti G, Montecucco A. (1999) The replication factory targeting sequence/PCNA-binding site is required in G(1) to control the phosphorylation status of DNA ligase I. EMBO J. 18:5745-54.
  8. Montecucco A, Rossi R, Levin DS, Gary R, Park MS, Motycka TA, Ciarrocchi G, Villa A, Biamonti G, Tomkinson AE. (1998) DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories. EMBO J. 17:3786-95.

 

 


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