Identification and functional analysis of PCNA1 and PCNA-like1 genes of Phaseolus coccineus

Abstract

Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and in many other processes in eukaryotic cells. Genetic analysis of Phaseolus coccineus showed the presence of at least two PCNA-like genes in the runner bean genome. Two PCNA genes have previously been found in a few plant species including Arabidopsis, tobacco, and maize. In these species, genes were nearly identical. Two cDNAs of P. coccineus PCNA (PcPCNA1 and PcPCNA-like1) have been identified that differ distinctly from each other. Interestingly, both the genetic organization of PcPCNA1 and PcPCNA-like1 genes and their expression patterns were similar, but these were the only similarities between these genes and their products. The identity between PcPCNA1 and PcPCNA-like1 at the amino acid level was only 54%, with PcPCNA-like1 lacking motifs that are crucial for the activity typical of PCNA. Consequently, these two proteins showed different properties. PcPCNA1 behaved like a typical PCNA protein: it formed a homotrimer and stimulated the activity of human DNA polymerase delta. In addition, PcPCNA1 interacted with a p21 peptide and was recognized by an anti-human PCNA monoclonal antibody PC10. By contrast, PcPCNA-like1 was detected as a monomer and was unable to stimulate the DNA polymerase delta activity. PcPCNA-like1 also could not interact with p21 and was not recognized by the PC10 antibody. Our results suggest that PcPCNA-like1 either is unable to function alone and therefore might be a component of the heterotrimeric PCNA ring or may have other, yet unknown functions. Alternatively, the PcPCNA-like1 gene may represent a pseudogene.

Fig. 1.

Alignment of amino acid sequences of PcPCNA1 (accession number: ABQ96591) and PcPCNA-like1 (accession number: ABQ96593) proteins. The analysis was done using StretcherP and the graphical representation was made using GeneDoc software. Identical amino acids are denoted as white letters on a black background. The black letters on grey and white backgrounds represent different amino acids. The residue D⁴¹ and motifs I (H¹²⁵L¹²⁶G¹²⁷I¹²⁸), II (V¹⁸⁸D¹⁸⁹K¹⁹⁰), and III (L²⁵¹A²⁵²P²⁵³K²⁵⁴) are marked. Dashes represent gaps introduced to maximize identities.

Fig. 2.

Southern blot and PCR analysis of P. coccineus genomic DNA. (A) Structure of PcPCNA1 and PcPCNA-like1 genes. The nucleotide sequences were analysed using WebGene software. Exons (E), introns (I), and 5′-UTR and 3′-UTR regions are marked. The border sequences of introns termini are placed in the boxes. The positions of internal sites recognized by restriction enzymes used in the Southern blotting analysis are marked. (B) Southern blotting results. 30 μg of the genomic DNA isolated from P. coccineus seedlings were digested with BamHI (lanes 2 and 8), BglII (lanes 3 and 9), EcoRI (lanes 4 and 10), HindIII (lanes 5 and 11) or XbaI (lanes 6 and 12), separated in 0.8% agarose gel and subjected to Southern blot procedure with the PcPCNA1 (lanes 2–6) or PcPCNA-like1 (lanes 8–12) probe. Lanes 1 and 7: DNA molecular weight marker II Digoxigenin-labelled (Roche). Stars indicate position of DNA fragments detected with PcPCNA probes. (C) PCR results. PCR was performed using degenerate primers and gDNA isolated from P. coccineus seedlings (lane 3), or plasmid pTZ57R\T DNA containing genomic sequence of the PcPCNA1 gene (lane 1) or of the PcPCNA-like1 gene (lane 2). In negative control, DNA template was omitted (lane 4). Lane M: DNA size standards (1 kb DNA ladder).

Fig. 3.

PRINS analysis performed on metaphase chromosomes of P. coccineus. The reactions were performed with PcPCNA1 specific primers (A) and with PcPCNA-like1 specific primers (B) in the presence of DIG-12-dUTP and 3 units of Taq polymerase. After the reaction, slides were incubated with anti-DIG-fluorescein antibody and preparations were examined with the OLYMPUS BX60 Research System Microscope. The observed signals are denoted by white arrows. Each pictures shows a single diploid cell (2n=22).

Fig. 4.

Purification of the recombinant PcPCNA1 (A) and PcPCNA-like1 (B) proteins. The protein samples were separated on 12% SDS-PAGE gels stained with Coommassie dye. The level of production of PcPCNA1 and PcPCNA-like1 and the subsequent purification steps are shown. Lane 1: molecular mass marker; lanes 2 and 3: non-induced and induced BL21(DE3)[pET15bPcPCNA1] and BL21(DE3)[pET15bPcPCNA-like1]; lane 4: cell lyzates; lane 5: elution from nickel column; lane 6: heparin column flow-through; lane 7: elution from HiTrap Q HP sepharose (1 μg of purified protein).

Fig. 5.

Analysis of native structures of the PcPCNA1 and PcPCNA-like1 proteins. 250 μg of PcPCNA1, PcPCNA-like1, and AtPCNA2 protein were separately filtered through Superdex 200. The arrows indicate the retention time of proteins used as molecular mass standards: thyroglobulin (670 kDa), γ calf globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa).

Fig. 6.

Test for the stimulatory effect of PCNA proteins on DNA polymerase delta activity. The polymerase activity assay was performed using poly(dA)/oligo(dT) as a template, [³H]TTP and 0.54 units of human DNA polymerase delta either alone or in the presence of 5 μg of the tested proteins: human PCNA (HsPCNA), PcPCNA1, PcPCNA-like1 or BSA (negative control). In addition, these proteins were assayed in the absence of human DNA polymerase delta. The radioactivity of the acid-precipitable material, measured in cmp, was then converted to pmol of [³H]TMP incorporated during 30 min of the reaction.

Fig. 7.

Analysis of PcPCNA1 and PcPCNA-like1 binding to p21 peptide. Biotinylated p21 peptide was attached to streptavidin–agarose beads and incubated with 1 μg of the recombinant protein followed by extensive washing. The samples were then separated in 12% SDS-PAGE and stained with Coommassie dye. Lane 1: molecular mass marker; lane 2: streptavidin–agarose beads without p21, reacted with PcPCNA1; lane 3: beads with p21, reacted with PcPCNA1; lane 4: beads without p21, reacted with PcPCNA-like1; lane 5: beads with p21 reacted with PcPCNA-like1; lane 6: beads without p21, reacted with HsPCNA; lane 7: beads with p21, reacted with HsPCNA.

Fig. 8.

Western blot analysis of PcPCNA proteins with anti-human PCNA monoclonal antibody (PC10). One microgram of PcPCNA1, PcPCNA-like1, and HsPCNA was separated in 12% polyacrylamide gel and subjected to Western blot analysis using PC10 antibodies. Lane 1: PcPCNA1; lane 2: PcPCNA-like1; lane 3: HsPCNA. The positions of molecular mass markers are indicated.

Fig. 9.

Real-time RT-PCR analysis of PcPCNA genes expression. (A) Relative changes in the PcPCNA1 and PcPCNA-like1 transcript levels during the early stages of germination from 0 h to 96 h. (B) Relative expression of PcPCNA1 and PcPCNA-like1 genes in P. coccineus root, stem, leaf, and micropylum. The figure presents data obtained in one of three independent experiments, and is representative for the observed changes.

Fig. 10.

Phylogenetic tree of PCNA constructed by the Neighbor–Joining method based on amino acid (aa) sequences from P. coccineus and other selected eukaryotic organisms was created using MEGA 3.1 software (Kumar et al., 2004). The scale bar represents 0.05 substitutions per site, and the numbers next to the nodes are bootstrap values from 100 000 replicates. Values equal to or higher than 80% are shown.