Giovanni Ciarrocchi

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

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF‐C p140) have a homologous sequence of ∼20 amino acids at their N‐termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N‐terminal fragments of DNA ligase I and the RF‐C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF‐C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA‐binding motif have been identified in other DNA replication enzymes and in p21CIP1/WAF1, we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

Determination of pyrimidine dimer unwinding angle by measurement of DNA electrophoretic mobility

Abstract The unwinding angle produced by the formation of one pyrimidine dimer has been estimated to be −14.3 ° ±0.2 °. This value has been obtained by titrating the number of pyrimidine dimers necessary to reduce the number of superhelical turns by one in each topoisomer obtained by treatment of a supercoiled DNA with DNA topoisomerase I.

The replication factory targeting sequence/PCNA- binding site is required in G 1 to control the phosphorylation status of DNA ligase I

The recruitment of DNA ligase I to replication foci in S phase depends on a replication factory targeting sequence that also mediates the interaction with proliferating cell nuclear antigen (PCNA) in vitro. By exploiting a monoclonal antibody directed at a phospho‐epitope, we demonstrate that Ser66 of DNA ligase I, which is part of a strong CKII consensus site, is phosphorylated in a cell cycle‐dependent manner. After dephosphorylation in early G1, the level of Ser66 phosphorylation is minimal in G1, increases progressively in S and peaks in G2/M phase. The analysis of epitope‐tagged DNA ligase I mutants demonstrates that dephosphorylation of Ser66 requires both the nuclear localization and the PCNA‐binding site of the enzyme. Finally, we show that DNA ligase I and PCNA interact in vivo in G1 and S phase but not in G2/M. We propose that dephosphorylation of Ser66 is part of a novel control mechanism to establish the pre‐replicative form of DNA ligase I.

The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication

DNA replication in mammalian cells occurs in discrete nuclear foci called ‘replication factories’. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non‐S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)‐DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N‐terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub‐nuclear compartmentalization.

Control of DNA Replication and Cell Proliferation in Eukaryotes by Aphidicolin

The mycotoxin aphidicolin specifically inhibits nuclear DNA synthesis in eukaryotic cells by intereacting with the replicative DNA polymerase a. The drug does not bind directly to DNA nor does it interfere with RNA, protein and nucleic acid precursor synthesis. Aphidicolin offers a new tool for analyzing the mechanism of DNA replication and repair in eukaryotes and for studying the role of eukaryotic DNA polymerases. The drug might also be valuable therapeutically for controlling excessive cell proliferation without adverse effects upon non-multiplying cells. We describe here how to synchronize DNA synthesis in human cells by a double block with aphidicolin and the properties of two human cell lines resistant to the drug.

Induction of polynucleotide ligase in human lymphocytes stimulated by phytohemoagglutinin

Abstract The treatment of human lymphocytes with phytohemoagglutinin causes the appearance of the activity of polynucleotide ligase, rising at least 50 fold from levels below the background. This increase takes place in the fourth or fifth day after treatment, and is delayed by one day approximately with respect to the rise of DNA synthesis rate; the activity of two other enzymes of DNA metabolism, DNA polymerase and a DNase acting on single-stranded DNA, increases in parallel with the DNA synthesis rate.

On the identity of dnaP and dnaF genes of Bacillus subtilis

SummaryThe dnaP strains of Bacillus subtilis are altered in the initiation of DNA replication at high temperature (Riva et al., 1975). Fine mapping of the gene shows that it is located very close to the dnaF gene, described by Karamata and Gross (1970) and mapped by Love et al. (1976) in the polC region. The phenotype of both mutants is indistinguishable: the DNA synthesis stops at non permissive temperature after synthesizing an amount of DNA equivalent to the completion of the rounds of replication already initiated; at permissive temperature they are abnormally sensitive to MMS and are reduced in the ability to be transformed. Both mutants are to be considered as belonging to the dnaF locus.The dnaF gene is very close to the polC gene, which specifies the DNA polymerase III of B. subtilis. The DNA polymerase III of the dnaF mutants is not temperature sensitive in vitro, however, the level of this enzyme is lower by a factor of 4 or 5 in the dnaF mutants, at the permissive temperature. Following shift of dnaF cultures to the non permissive temperature, the level of DNA polymerase III activity specifically decreases further by a factor of at least 10 in the mutant, whereas the DNA polymerase I level is unaffected.The possible roles of the dnaF gene in the control of the cellular level of the DNA polymerase III, and the possibility of a regulatory role of DNA polymerase III in the initiation of DNA replication in bacteria are discussed.

Cloning and sequence analysis of a cDNA coding for the murine DNA ligase I enzyme

A complementary DNA (2961 bp) containing the complete coding sequence for murine DNA ligase I was isolated from a mouse fibroblast cDNA library using a cDNA encoding the human protein as a probe. An open reading frame of 2748 bp, encoding a protein of 916 amino acids (aa), was identified. Northern blot analysis of total RNA extracted from mouse fibroblasts showed a single band with a mobility corresponding to a size of 3.2 kb whose level increases upon serum stimulation of quiescent mouse NIH-3T3 cells. Alignment of the murine and human deduced aa sequences showed an overall 83% identity, that rises to 91% if only the sequence on the C-terminal portion of the protein containing the active site is considered.

Multiple roles of DNA ligase at the replication fork

The loss of superhelical turns from a covalently closed duplex DNA exposed to bacteriophage T4 DNA ligase in the presence of AMP and Mg2+ has recently been found to be gradual and not sudden (Montecucco, A. and Ciarrocchi, G. (1988) Nucleic Acids Res. 16, 7369-7381). In this paper, we show that the AMP-dependent DNA relaxation catalyzed by human and E. coli DNA ligases also takes place according to a step-wise mechanism. DNA relaxation is inhibited by pyrophosphate, by ATP (or NAD in the case of the E. coli enzyme) and by high ionic strength and is essentially distributive with the human or T4 DNA ligases, and processive with the bacterial one. The AMP-dependent ability of DNA ligases to relax DNA might allow these enzymes to relieve possible topological complications of the nascent double helix generated by the replication of the lagging strand.

Lack of discrimination between DNA ligases I and III by two classes of inhibitors, anthracyclines and distamycins

We have measured the effects of eight distamycin and two anthracycline derivatives on polynucleotide joining and self-adenylating activities of human DNA ligase I and rat DNA ligases I and III. All test drugs show good inhibitory activity against the three enzymes in the poly[d(A-T)] joining assay. Several distamycins also inhibit the DNA-independent self-adenylation reaction catalysed by the human enzyme and, to a lesser extent, by rat DNA ligases. These results confirm that anthracyclines and distamycins express their inhibitory action against DNA joining activities mainly via specific interactions with the substrate, and suggest that the three test DNA ligases utilize similar, if not identical, mechanisms of recognition and interaction with DNA-drug complexes. Our findings also indicate that distamycins have a greater affinity for human DNA ligase I than for rat enzymes, suggesting that, in this respect, rat DNA ligase I is more similar to rat DNA ligase III than to human DNA ligase I.