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Thermostable bacterial polymerases like Taq, Therminator and Vent exo(-) are able to perform DNA synthesis by using modified DNA precursors, a property that is exploited in several therapeutic and biotechnological applications. Viral polymerases are also known to accept modified substrates, and this has proven crucial in the development of antiviral therapies. However, non-thermostable polymerases of bacterial origin, or engineered variants, that have similar substrate tolerance and could be used for synthetic biology purposes remain to be identified. We have identified the α subunit of Escherichia coli polymerase III (Pol III α) as a bacterial polymerase that is able to recognise and process as substrates several pyrophosphate-modified dATP analogues in place of its natural substrate dATP for template-directed DNA synthesis. A number of dATP analogues featuring a modified pyrophosphate group were able to serve as substrates during enzymatic DNA synthesis by Pol III α. Features such as the presence of potentially chelating chemical groups and the size and spatial flexibility of the chemical structure seem to be of major importance for the modified leaving group to play its role during the enzymatic reaction. In addition, we could establish that if the pyrophosphate group is altered, deoxynucleotide incorporation proceeds with an efficiency varying with the nature of the nucleobase. Our results represent a great step towards the achievement of a system of artificial DNA synthesis hosted by E. coli and involving the use of altered nucleotide precursors for nucleic acid synthesis. ispartof: Chembiochem vol:13 issue:16 pages:2439-44 ispartof: location:Germany status: published

International audience; Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, that displays both redox and chaperone properties. Since three out of the six proteins of the YbbN interactome (Butland et al., 2005) are components of DNA polymerase 3 holoenzyme (i.e. the β-clamp DnaN, the θ subunit HolE and the δ' subunit HolB), we investigated whether the ybbN mutant presents DNA replication defects. We found that this mutant incorporates (3)H-thymidine at higher rates than the parental strain and displays overinitiation, hypermutator and filamentation phenotypes with the occurrence of anucleated cells. Moreover, YbbN functions as a bona fide chaperone in the refolding of the urea-unfolded β-clamp. These results suggest that the DNA replication and cell division defects of the ybbN mutant might best be explained by chaperone functions of YbbN in the biogenesis of DNA polymerase 3 holoenzyme.

International audience; Of the four subunits constituting DNA polymerase delta, subunit C or p66 has been shown to mainly mediate polymerase interaction with PCNA, an auxiliary factor that greatly enhances DNA polymerase delta processivity on primed DNA templates. Here, we provide evidence that a highly conserved region located between amino acids 384 and 399 in the C-terminus of p66 is phosphorylated, most probably by Protein kinase CK2, and that another region, most probably located within the PCNA interacting domain in its extreme C-terminus, regulates its interaction with PCNA. Phosphorylation of p66 is associated with its co-localization with large subunit of DNA polymerase delta, p125, and PCNA, to the insoluble chromatin fraction at the beginning of S-phase. Taken together, the results provide evidence that concurrent phosphorylation events in p66 may positively and negatively regulate its activity and interactions with other components of the replisome during the cell cycle.

Inactivated replication forks may be reversed by the annealing of leading- and lagging-strand ends, resulting in the formation of a Holliday junction (HJ) adjacent to a DNA double-strand end. In Escherichia coli mutants deficient for double-strand end processing, resolution of the HJ by RuvABC leads to fork breakage, a reaction that we can directly quantify. Here we used the HJ-specific resolvase RusA to test a putative role of the RuvAB helicase in replication fork reversal (RFR). We show that the RuvAB complex is required for the formation of a RusA substrate in the polymerase III mutants dnaEts and holD, affected for the Pol III catalytic subunit and clamp loader, and in the helicase mutant rep. This finding reveals that the recombination enzyme RuvAB targets forks in vivo and we propose that it directly converts forks into HJs. In contrast, RFR occurs in the absence of RuvAB in the dnaNts mutant, affected for the processivity clamp of Pol III, and in the priA mutant, defective for replication restart. This suggests alternative pathways of RFR.

Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design.

International audience; Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication.

International audience; We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase delta. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2+/rhp9+. This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30 degrees C, but were defective in this checkpoint function when treated with MMS at 37 degrees C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37 degrees C. Furthermore, the crb2+ gene was required, together with the cds1+ gene, for the S/M checkpoint at 30 degrees C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the poldeltats3 background at both 30 degrees C and 37 degrees C. The rapid death phenotype was independent of the checkpoint functions.

International audience; DNA replication and DNA repair are essential cell cycle steps ensuring correct transmission of the genome. The feedback replication control system links mitosis to completion of DNA replication and partially overlaps the radiation checkpoint control. Deletion of the chk1/rad27 gene abolishes the radiation but not the replication feedback control. Thermosensitive mutations in the DNA polymerase delta, cdc18 or cdc20 genes lead cells to arrest in the S phase of the cell cycle. We show that strains carrying any of these mutations enter lethal mitosis in the absence of the radiation checkpoint chk1/rad27. We interpret these data as an indication that an assembled replisome is essential for replication dependent control of mitosis and we propose that the arrest of the cell cycle in the thermosensitive mutants is due to the chk1+/rad27+ pathway, which monitors directly DNA for signs of damage.

International audience; We have investigated the expression of two Schizosaccharomyces pombe replicative DNA polymerases alpha and delta during the cell cycle. The pol alpha+ and pol delta+ genes encoding DNA polymerases alpha and delta were isolated from S. pombe. Both pol alpha+ and pol delta+ genes are single copy genes in haploid cells and are essential for cell viability. In contrast to Saccharomyces cerevisiae homologs, the steady-state transcripts of both S. pombe pol alpha+ and pol delta+ genes were present throughout the cell cycle. Sequence analysis of the pol alpha+ and pol delta+ genes did not reveal the Mlu I motifs in their upstream sequences that are involved in cell cycle-dependent transcription of S. cerevisiae DNA synthesis genes as well as the S. pombe cdc22+ gene at the G1/S boundary. However, five near-match Mlu I motifs were found in the upstream region of the pol alpha+ gene. S. pombe DNA polymerases alpha and delta proteins were also expressed constantly throughout the cell cycle. In addition, the enzymatic activity of the S. pombe DNA polymerase alpha measured by in vitro assay was detected at all stages of the cell cycle. Thus, these S. pombe replicative DNA polymerases, like that of S. pombe cdc17+ gene, are expressed throughout the cell cycle at the transcriptional and protein level. These results indicate that S. pombe has at least two regulatory modes for the expression of genes involved in DNA replication and DNA precursor synthesis.

International audience; DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.