Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.     

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda

DNA polymerase λ (pol λ) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol λ either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol λ had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn⁺⁺ and Mg⁺⁺ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol λ; (iii) pol λ preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol λ, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol λ and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol λ are discussed.     

The effect of coating single- and double-stranded DNA with the recombinase A protein of Escherichia coli on transgene integration in mice

Embryo survival and transgene integration rates are two major factors that influence the efficiency of transgenic animal production by pronuclear microinjection. Recombinase A protein-coated transgenes were compared for transgene integration and embryo survival with their non-coated counterparts in both single- and double-stranded forms. Murine zygotes were microinjected with a large 30 kb α_S1-casein/human lysozyme DNA construct and a small 5.5 kb β-lactoglobulin/desaturase DNA construct using four different construct preparations for each gene. The preparations included recombinase A protein-coated, single- and double-stranded DNA constructs and non-coated, single- and double-stranded DNA constructs. Using conventional non-coated, double-stranded DNA constructs, we obtained a transgene integration efficiency of 1.5% (1352 embryos transferred produced 20 transgenic pups). The same double-stranded DNA constructs coated with recombinase A protein yielded a similar percentage of transgene integration (1.1%, 18/1697). Using single-stranded DNA, non-coated constructs produced a transgene integration rate of 0.5%, while none of the 1040 zygotes injected with recombinase A-coated constructs produced transgenic pups. While recombinase A protein coating produced no effect on embryo survival, litter size or pregnancy rate with double-stranded constructs, a detrimental effect was observed on embryo survival (P < 0.001) and pregnancy rate (P < 0.005) with recombinase A protein coating of single-stranded human lysozyme DNA constructs. A trend toward increased embryo survival (P = 0.054) with no difference in pregnancy rate (P > 0.05) was observed with the recombinase A protein coating of single-stranded desaturase constructs. These results suggest that recombinase A protein coating of single- and double-stranded DNA constructs produced no significant differences (P > 0.05) in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born.     

Genetic diversity of the great bustard in Iberia and Morocco: risks from current population fragmentation

We studied the genetic diversity of great bustards (Otis tarda) in Iberia and Morocco, the main stronghold of this globally endangered species. Samples were collected from 327 individuals covering most of the distribution range within the study area. Sequence variation in a 657 bp fragment of the mtDNA control region revealed 20 variable sites defining 22 haplotypes, two of them exclusive to Morocco. Genetic diversity showed marked regional differences (π = 0–0.53, h = 0–0.89). Multidimensional scaling analysis based on F _ST values showed a clear division between Morocco and the Iberian Peninsula, with no evidence of current gene flow between them. Our results suggest that Morocco, where few matrilines have persisted to present, was colonized from Iberia thousands of years ago. Last century reports suggest dispersal through Gibraltar, when the species was more abundant at both sides of the Strait but later population declines and the Strait’s barrier effect have favoured current genetic isolation. Within Iberia, only the most peripheral populations (Navarra, Aragón and Andalusia) differed significantly from the main ones in central Spain. The first two showed extremely low genetic diversity and are probably threatened by inbreeding depression. Diversity was higher in Andalusia, where three exclusive haplotypes were found, suggesting some degree of isolation from other populations. Andalusia and Morocco could be regarded as separate management units which hold a significant proportion of the current genetic diversity and thus deserve urgent conservation measures.     

Sexual Traits as Quality Indicators in Lekking Male Great Bustards

We studied the development of two sexual traits, whiskers and neck plumage, in relation to sexual selection in 41 free‐living great bustard, Otis tarda, males radio‐tracked at nine leks in central Spain in 1998–2001. During the pre‐breeding male–male competition period (Feb.) prior to female arrival, number and length of whiskers correlated with weight, but not with body size or age. Whiskers may thus have evolved as an intrasexual indicator of weight, which in the absence of other weapons in this species is decisive in male–male combats. Signalling through whiskers contributes to minimizing dangerous aggressive interactions in the lek. During the mating period (Apr.), both whisker and neck development were correlated with weight and age. Males reaching higher expression of both traits exhibited higher display intensity, a more prolonged display period through the mating season, and a higher estimated mating success. Moreover, interannual changes in a male’s expression of both traits were associated with changes in its display intensity and estimated mating success. Our results resolve earlier debates and contradictory results from previous authors, suggesting that these two secondary sexual traits, whiskers and neck, may function as reliable indicators of age and weight, the two main factors determining social rank of males in great bustard leks, during both rival assessment and mate choice. Their dual functions provide support for the pre‐existing trait and redundant signal hypotheses and suggest that multiple ornaments functioning as redundant signals might be more widespread than previously acknowledged.     

Dual Src and Abl inhibitors target wild type Abl and the AblT315I Imatinib-resistant mutant with different mechanisms

The tyrosine kinase Src and its close homolog Abl, both play important roles in chronic myelogenous leukemia (CML) progression and Imatinib resistance. No clinically approved inhibitors of the drug-resistant AblT315I exist to date. Here, we present a thorough kinetic analysis of two potent dual Src-Abl inhibitors towards wild type Src and Abl, and the AblT315I mutant. Our results show that the most potent compound BO1 shows only a modest loss of potency (fourfold) towards the AblT315I mutant in vitro and was an ATP-competitive inhibitor of wild type Abl but it acted as a non-competitive inhibitor in the case of AblT315I.     

Towards novel S-DABOC inhibitors: synthesis, biological investigation, and molecular modeling studies

A small family of S-DABO cytosine analogs (S-DABOCs) has been synthesized and biologically evaluated as HIV-1 inhibitor both on wild type (wt) and drug-resistant mutants leading to the identification of an interesting compound (5d). Molecular modeling studies have been finally performed in order to rationalize the results.