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Biomedical subjects

Arturo Falaschi

Publications and source records attributed to Arturo Falaschi.

9 recordsLinked to original sources
PubMed2004

Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A.

RecQ helicases are required for the maintenance of genome stability. Characterization of the substrate specificity and identification of the binding partners of the five human RecQ helicases are essential for understanding their function. In the present study, we have developed an efficient baculovirus expression system that allows us to obtain milligram quantities of recombinant RECQ1. Our gel filtration and dynamic light scattering experiments show that RECQ1 has an apparent molecular mass of 158 kDa and a hydrodynamic radius of 5.4 +/- 0.6 nm, suggesting that RECQ1 forms dimers in solution. The oligomeric state of RECQ1 remains unchanged upon binding to a single-stranded (ss)DNA fragment of 50 nt. We show that RECQ1 alone is able to unwind short DNA duplexes (<110 bp), whereas considerably longer substrates (501 bp) can be unwound only in the presence of human replication protein A (hRPA). The same experiments with Escherichia coli SSB show that RECQ1 is specifically stimulated by hRPA. However, hRPA does not affect the ssDNA-dependent ATPase activity of RECQ1. In addition, our far western, ELISA and co-immunoprecipitation experiments demonstrate that RECQ1 physically interacts with the 70 kDa subunit of hRPA and that this interaction is not mediated by DNA.

Adenosine Triphosphatases↗
PubMed2004

Modular structure of the human lamin B2 replicator.

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Base Sequence↗
PubMed2003

Localization of proteins bound to a replication origin of human DNA along the cell cycle.

The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site.

Base Sequence↗
PubMed2003

In vitro protein-DNA interactions at the human lamin B2 replication origin.

The complexity of mammalian origins of DNA replication has prevented, so far, the in vitro studies of the modalities of initiator protein binding and origin selection. We approached this problem by utilizing the human lamin B2 origin, wherein the precise start sites of replication initiation have been identified and known to be bound in vivo by the origin recognition complex (ORC). In order to analyze the in vitro interactions occurring at this origin, we have compared the DNA binding requirements and patterns of the human recombinant Orc4 with those of preparations of HeLa nuclear proteins containing the ORC complex. Here we show that both HsOrc4 alone and HeLa nuclear proteins recognize multiple sites within a 241-bp DNA sequence encompassing the lamin B2 origin. The DNA binding activity of HeLa cells requires the presence of ORC and can be reproduced in the absence of all the other proteins known to be recruited to origins by ORC. Both HsOrc4 alone and HeLa nuclear proteins exhibit cooperative and ATP-independent binding. This binding covers nucleotides 3853-3953 and then spreads outward. Because this region contains the start sites of DNA synthesis as well as the area protected in vivo and preserves protein binding capacity in vitro after removal of a fraction of the protected region, we suggest that it could contain the primary binding site. Thus the in vitro approach points to the sequence requirements for ORC binding as a key element for origin recognition.

Adenosine Triphosphate↗
PubMed2003

Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression.

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.

Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed2003

Is DNA sequence sufficient to specify DNA replication origins in metazoan cells?

DNA replication occupies a central position in the cell cycle and, therefore, in the development and life of multicellular organisms. During the last 10 years, our comprehension of this important process has considerably improved. Although the mechanisms that coordinate DNA replication with the other moments of the cell cycle are not yet fully understood, it is known that they mainly operate through DNA replication origins and the protein complexes bound to them. In eukaryotes, the packaging status of chromatin seems to be part of the mechanism that controls whether or not and when during the S-phase a particular origin will be activated. Intriguingly, the protein complexes bound to DNA replication origins appear to be directly involved in controlling chromatin packaging. In this manner they can also affect gene expression. In this review we focus on DNA replication origins in metazoan cells and on the relationship between these elements and the structural and functional organization of the genome.

Animals↗
PubMed2002

Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human replication protein A.

The RecQ helicases are involved in several aspects of DNA metabolism. Five members of the RecQ family have been found in humans, but only two of them have been carefully characterized, BLM and WRN. In this work, we describe the enzymatic characterization of RECQ1. The helicase has 3' to 5' polarity, cannot start the unwinding from a blunt-ended terminus, and needs a 3'-single-stranded DNA tail longer than 10 nucleotides to open the substrate. However, it was also able to unwind a blunt-ended duplex DNA with a "bubble" of 25 nucleotides in the middle, as previously observed for WRN and BLM. We show that only short DNA duplexes (<30 bp) can be unwound by RECQ1 alone, but the addition of human replication protein A (hRPA) increases the processivity of the enzyme (>100 bp). Our studies done with Escherichia coli single-strand binding protein (SSB) indicate that the helicase activity of RECQ1 is specifically stimulated by hRPA. This finding suggests that RECQ1 and hRPA may interact also in vivo and function together in DNA metabolism. Comparison of the present results with previous studies on WRN and BLM provides novel insight into the role of the N- and C-terminal domains of these helicases in determining their substrate specificity and in their interaction with hRPA.

Adenosine Triphosphatases↗
PubMed2002

Studies on the mode of Ku interaction with DNA.

The Ku heterodimer plays a central role in non-homologous end-joining. The binding of recombinant Ku to DNA has been investigated by dynamic light scattering, double-filter binding, fluorescence spectroscopy, and band shift assays. The hydrodynamic radius of Ku in solution is 5.2 nm and does not change when a 25-bp double-strand DNA (dsDNA) fragment (D25) is added, indicating that only one Ku molecule binds to a 25-bp fragment. The dissociation constant (k(d)) for the binding to D25 is 3.8 +/- 0.9 nm. If both ends of the substrate are closed with hairpin loops, Ku is still able to bind with little change in the k(d). The k(d) is not affected by ATP, Mg(2+), or ionic strength. However, the addition of bovine serum albumin decreases the k(d) by 2-fold. DNA substrates of 50 bp can bind two Ku molecules, whereas three molecules are bound to a 75-bp substrate. Data analysis with the Hill equation yields a value of the Hill coefficient (n) close to 1, and the k(d) values for the binding of Ku to both ends of these substrates are the same. Thus, we demonstrate that there is no cooperative interaction among the Ku heterodimers binding longer substrates.

Adenosine Triphosphate↗