PubMed Health⌕ Search

Biomedical subjects

Domenico Maiorano

Publications and source records attributed to Domenico Maiorano.

10 recordsLinked to original sources
PubMed2006

A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus.

Initiation of DNA synthesis involves the loading of the MCM2-7 helicase onto chromatin by Cdt1 (origin licensing). Geminin is thought to prevent relicensing by binding and inhibiting Cdt1. Here we show, using Xenopus egg extracts, that geminin binding to Cdt1 is not sufficient to block its activity and that a Cdt1-geminin complex licenses chromatin, but prevents rereplication, working as a molecular switch at replication origins. We demonstrate that geminin is recruited to chromatin already during licensing, while bulk geminin is recruited at the onset of S phase. A recombinant Cdt1-geminin complex binds chromatin, interacts with the MCM2-7 complex and licenses chromatin once per cell cycle. Accordingly, while recombinant Cdt1 induces rereplication in G1 or G2 and activates an ATM/ATR-dependent checkpoint, the Cdt1-geminin complex does not. We further demonstrate that the stoichiometry of the Cdt1-geminin complex regulates its activity. Our results suggest a model in which the MCM2-7 helicase is loaded onto chromatin by a Cdt1-geminin complex, which is inactivated upon origin firing by binding additional geminin. This origin inactivation reaction does not occur if only free Cdt1 is present on chromatin.

Animals↗
PubMed2006

MCM proteins and DNA replication.

The MCM proteins identify a group of ten conserved factors functioning in the replication of the genomes of archae and eukaryotic organisms. Among these, MCM2-7 proteins are related to each other and form a family of DNA helicases implicated at the initiation step of DNA synthesis. Recently this family expanded by the identification of two additional members that appear to be present only in multicellular organisms, MCM8 and MCM9. The function of MCM8 is distinct from that of MCM2-7 proteins, while the function of MCM9 is unknown. MCM1 and MCM10 are not related to this family, nor to each other, but also function in DNA synthesis.

Animals↗
PubMed2005

Identification of full genes and proteins of MCM9, a novel, vertebrate-specific member of the MCM2-8 protein family.

MCM2-7 proteins are conserved replication factors functioning as DNA helicases during DNA synthesis. MCM8 is another member of this family, which appears to be specific for higher eukaryotes, as it is absent in worms and yeast. Here we report the complete identification of a novel member of this family, the MCM9 protein. Like MCM8, MCM9 is only present in the genome of higher eukaryotes. This protein contains an MCM8-like ATP binding and hydrolysis motif implicated in helicase activity. Strikingly, MCM9 also contains a unique carboxy-terminal domain which has only weak homology to MCM2-7 and MCM8 but is conserved within MCM9 homologs. We also show that the very recently reported human MCM9 protein (HsMCM9), which resembles a truncated MCM-like protein missing a part of the MCM2-7 signature domain, is an incomplete form of the full length HsMCM9 described here. Searching the human genome with either the newly identified human MCM9 or other MCM protein sequences, we did not detect further additional members of this DNA helicase family and suggest that it is constituted of eight members, falling into two different groups, one constituted by the MCM2-7 complex and the other by MCM8 and MCM9, which are present only in higher eukaryotes.

Amino Acid Sequence↗
PubMed2005

MCM8 is an MCM2-7-related protein that functions as a DNA helicase during replication elongation and not initiation.

MCM2-7 proteins are replication factors required to initiate DNA synthesis and are currently the best candidates for replicative helicases. We show that the MCM2-7-related protein MCM8 is required to efficiently replicate chromosomal DNA in Xenopus egg extracts. MCM8 does not associate with the soluble MCM2-7 complex and binds chromatin upon initiation of DNA synthesis. MCM8 depletion does not affect replication licensing or MCM3 loading but slows down DNA synthesis and reduces chromatin recruitment of RPA34 and DNA polymerase-alpha. Recombinant MCM8 displays both DNA helicase and ATPase activities in vitro. Reconstitution experiments show that ATP binding in MCM8 is required to rescue DNA synthesis in MCM8-depleted extracts. MCM8 colocalizes with replication foci and RPA34 on chromatin. We suggest that MCM8 functions in the elongation step of DNA replication as a helicase that facilitates the recruitment of RPA34 and stimulates the processivity of DNA polymerases at replication foci.

Adenosine Triphosphatases↗
PubMed2005

Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts.

A crucial regulation for maintaining genome integrity in eukaryotes is to limit DNA replication in S phase to only one round. Several models have been proposed; one of which, the licensing model, predicted that formation of the nuclear membrane restricts access to chromatin to a positive replication factor. Cdt1, a factor binding to origins and recruiting the MCM2-7 helicase, has been identified as a component of the licensing system in Xenopus and other eukaryotes. Nevertheless, evidence is missing demonstrating a direct role for unscheduled Cdt1 expression in promoting illegitimate reinitiation of DNA synthesis. We show here that Xenopus Cdt1 is absent in G2 nuclei, suggesting that it might be either degraded or exported. Recombinant Cdt1, added to egg extracts in G2, crosses the nuclear membrane, binds to chromatin, and relicenses the chromosome for new rounds of DNA synthesis in combination with chromatin bound Cdc6. The mechanism involves rebinding of MCM3 to chromatin. Reinitiation is blocked by geminin only in G2 and is not stimulated by Cdc6, demonstrating that Cdt1, but not Cdc6, is limiting for reinitiation in egg extracts. These results suggest that removal of Cdt1 from chromatin and its nuclear exclusion in G2 is critical in regulating licensing and that override of this control is sufficient to promote illegitimate firing of origins.

Animals↗
PubMed2004

A hypophosphorylated form of RPA34 is a specific component of pre-replication centers.

Replication protein A (RPA) is a three subunit single-stranded DNA-binding protein required for DNA replication. In Xenopus, RPA assembles in nuclear foci that form before DNA synthesis, but their significance in the assembly of replication initiation complexes has been questioned. Here we show that the RPA34 regulatory subunit is dephosphorylated at the exit of mitosis and binds to chromatin at detergent-resistant replication foci that co-localize with the catalytic RPA70 subunit, at both the initiation and elongation stages of DNA replication. By contrast, the RPA34 phosphorylated form present at mitosis is not chromatin bound. We further demonstrate that RPA foci assemble on chromatin before initiation of DNA replication at sites functionally defined as initiation replication sites. Association of RPA with these sites does not require nuclear membrane formation, and is sensitive to the S-CDK inhibitor p21. We also provide evidence that RPA34 is present at initiation complexes formed in the absence of MCM3, but which contain MCM4. In such conditions, replication foci can form, and short RNA-primed nascent DNAs of discrete size are synthesized. These data show that in Xenopus, the hypophosphorylated form of RPA34 is a component of the pre-initiation complex.

Animals↗
PubMed2004

Crystal structure of the coiled-coil dimerization motif of geminin: structural and functional insights on DNA replication regulation.

We have determined the crystal structure of the coiled-coil domain of human geminin, a DNA synthesis inhibitor in higher eukaryotes. We show that a peptide encompassing the five heptad repeats of the geminin leucine zipper (LZ) domain is a dimeric parallel coiled coil characterized by a unique pattern of internal polar residues and a negatively charged surface that may target the basic domain of interacting partners. We show that the LZ domain itself is not sufficient to inhibit DNA synthesis but upstream and downstream residues are required. Analysis of a functional form of geminin by density sedimentation indicates an oligomeric structure. X-ray solution scattering experiments performed on a non-functional form of geminin having upstream basic residues and the LZ domain show a tetramer structure. Altogether, these results give a consistent identification and mapping of geminin interacting regions onto structurally important domains. They also suggest that oligomerization properties of geminin may be implicated in its inhibitory activity of DNA synthesis.

Amino Acid Sequence↗
PubMed2004

Specification of a DNA replication origin by a transcription complex.

In early Xenopus development, transcription is repressed and DNA replication initiates at non-specific sites. Here, we show that a site-specific DNA replication origin can be induced in this context by the assembly of a transcription domain. Deletion of the promoter element abolishes site-specific initiation, and its relocalization to an ectopic site induces a new origin of replication. This process does not require active transcription, and specification of the origin occurs mainly through a decrease in non-specific initiation at sites distant from the promoter. Finally, chromatin immunoprecipitation experiments suggest that site-specific acetylation of histones favours the selection of the active DNA replication origin. We propose that the specification of active DNA replication origins occurs by secondary epigenetic events and that the programming of chromatin for transcription during development contributes to this selection in higher eukaryotes.

Acetylation↗
PubMed2004

Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis.

Cdt1 is a conserved replication factor required in licensing the chromosome for a single round of DNA synthesis. The activity of Cdt1 is inhibited by geminin. The mechanism by which geminin interferes with Cdt1 activity is unknown. It is thought that geminin binds to and sequestrate Cdt1. We show that geminin does not interfere with the chromatin association of Cdt1 and that inhibition of DNA synthesis by geminin is observed following its accumulation on chromatin. The binding of geminin to chromatin has been investigated during S phase. We demonstrate that loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. We also show that geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes, which overlaps with the release of Cdt1. This regulation is strikingly different from that observed in somatic cells where the chromatin binding of these proteins is mutually exclusive. In contrast to somatic cells, we further show that geminin is stable during the early embryonic cell cycles. These results suggest a specific regulation of origin firing adapted to the rapid cell cycles of Xenopus and indicate that periodic degradation of geminin is not relevant to licensing during embryonic development.

Animals↗