Molecular characterisation of RecQ homologues in Arabidopsis thaliana - PubMed (original) (raw)

Molecular characterisation of RecQ homologues in Arabidopsis thaliana

F Hartung et al. Nucleic Acids Res. 2000.

Abstract

Members of the RecQ family of DNA helicases are involved in processes linked to DNA replication, DNA recombination and gene silencing. RecQ homologues of various animals have been described recently. Here, for the first time for plants, we characterised cDNAs of all in all six different RecQ-like proteins that are expressed to different extents in Arabidopsis thaliana. Surprisingly, three of these proteins are small in size [AtRecQl1, AtRecQl2, AtRecQl3-606, 705 and 713 amino acids (aa), respectively], whereas the two bigger proteins result from a duplication event during plant evolution [AtRecQl4A and AtRecQl4B-1150 and 1182 aa, respectively]. Another homologue (AtRecQsim, 858 aa) most probably arose by insertion of an unrelated sequence within its helicase domain. The presence of these homologues demonstrates the conservation of RecQ family functions in higher eukaryotes. We also detected a small gene (AtWRNexo) encoding 285 aa which, being devoid of any RecQ-like helicase domain, reveals a striking homology to the exonuclease domain of human Werner protein, a prominent RecQ helicase of larger size. By means of the two-hybrid assay we were able to detect an interaction between AtWRNexo and AtRecQl2, indicating that activities that reside in a single protein chain in mammals might in plants be complemented in trans.

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Figures

Figure 1

Figure 1

Schematic genomic structure of six RecQ-like homologues of A.thaliana. Introns are shown as white boxes within the grey shaded sequence. Introns with a conserved position throughout the six genes are shown as black boxes and are numbered above. The letters below the schematic sequence structure denote the most conserved amino acids of helicase domains II and V (see also Fig. 3).

Figure 2

Figure 2

Schematic structure of RecQ-like proteins from different organisms. The deduced proteins were aligned according to the helicase domains (shown as filled boxes). Stretches of acidic or basic amino acids are represented as light grey or dark grey boxes, respectively. The exonuclease domain of the human WRN protein is indicated. The size of the predicted proteins is given on the right.

Figure 3

Figure 3

Multiple alignment of the seven highly conserved helicase domains of RecQ-like proteins from A.thaliana and various other organisms. The multiple alignment was done with CLUSTALW (version 1.7; www.ibc.wustl.edu ). Gaps are represented by dashes, amino acids conserved in at least eight out of the ten sequences are grey shaded and the consensus amino acid is given in bold below the sequences. The helicase domains are numbered above the sequences. Amino acids considered as conserved in a given position are: (D,E); (K,R); (I,L and V). The organisms included in the alignment are, from top to bottom: E.coli; A.thaliana; N.crassa; H.sapiens; M.musculus and S.cerevisiae.

Figure 4

Figure 4

Southern analysis of the A.thaliana members of the RecQ family. The indicated fragment sizes are calculated from the sequence information of the corresponding genomic BAC clones. All genes show a banding pattern that is in accordance with a presumed single copy status in the Arabidopsis genome.

Figure 5

Figure 5

The small WRNexo gene from A.thaliana. (A) Schematic intron/exon structure of the gene. The five introns are represented as white boxes in the grey shaded sequence. (B) Alignment of AtWRNexo with the N-terminus of the WRN protein from mouse and the X.laevis FFA protein. Amino acids conserved in all three positions are given as letters below the sequence. Highly conserved amino acid stretches are represented as grey shaded boxes. Amino acids considered as conserved in a given position are: (D,E); (K,R); (I,L and V).

Figure 6

Figure 6

Two-hybrid analysis to detect interactions of AtWRNexo with AtRecQl1 to 3. AtWRNexo and AtRecQl2 showed a strong interaction after 4 h X-gal staining (left). The panels on the right with AtRecQl1 or 3 and AtWRNexo were X-gal stained for 12 h without resulting in a positive signal. As a control the SNF1 + 4 interaction is given in the upper part of the figure.

Figure 7

Figure 7

Comparative RT–PCR of AtRecQl1 to 4B and AtWRNexo using mRNA from different tissues (A) and a quantification of expression in flowers (B). (A) Each cDNA was amplified with 38 PCR cycles starting with reverse transcription of 10 ng mRNA from Arabidopsis rosette leaves, shoots, flowers or seedlings. (B) Each cDNA was amplified with 35 PCR cycles starting with reverse transcription of 10 ng mRNA from Arabidopsis flowers or a 1:10 or 1:100 dilution of this RT, respectively. (A and B) One-fifth of the PCR was subjected to gel electrophoresis and EtBr staining.

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