BackgroundNAC (NAM, ATAF1/2 and CUC2) domain proteins are plant-specific transcriptional factors known to play diverse roles in various plant developmental processes. NAC transcription factors comprise of a large gene family represented by more than 100 members in Arabidopsis, rice and soybean etc. Recently, a preliminary phylogenetic analysis was reported for NAC gene family from 11 plant species. However, no comprehensive study incorporating phylogeny, chromosomal location, gene structure, conserved motifs, and expression profiling analysis has been presented thus far for the model tree species Populus.ResultsIn the present study, a comprehensive analysis of NAC gene family in Populus was performed. A total of 163 full-length NAC genes were identified in Populus, and they were phylogeneticly clustered into 18 distinct subfamilies. The gene structure and motif compositions were considerably conserved among the subfamilies. The distributions of 120 Populus NAC genes were non-random across the 19 linkage groups (LGs), and 87 genes (73%) were preferentially retained duplicates that located in both duplicated regions. The majority of NACs showed specific temporal and spatial expression patterns based on EST frequency and microarray data analyses. However, the expression patterns of a majority of duplicate genes were partially redundant, suggesting the occurrence of subfunctionalization during subsequent evolutionary process. Furthermore, quantitative real-time RT-PCR (RT-qPCR) was performed to confirm the tissue-specific expression patterns of 25 NAC genes.ConclusionBased on the genomic organizations, we can conclude that segmental duplications contribute significantly to the expansion of Populus NAC gene family. The comprehensive expression profiles analysis provides first insights into the functional divergence among members in NAC gene family. In addition, the high divergence rate of expression patterns after segmental duplications indicates that NAC genes in Populus are likewise to have been retained by substantial subfunctionalization. Taken together, our results presented here would be helpful in laying the foundation for functional characterization of NAC gene family and further gaining an understanding of the structure-function relationship between these family members.

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.

The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection of specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.Each time that a eukaryotic cell divides, it replicates its genome once and only once. In the budding yeast Saccharomyces cerevisiae, the mechanism that regulates this process begins with assembly of a single six-subunit origin recognition complex (ORC) at specific sites along the genome and DNA synthesis begins at or near the site where ORC binds (4). In S. cerevisiae, these DNA replication origins consist of 100 to 150 bp that can act as autonomously replicating sequences (ARS) and contain several noninterchangeable elements (34a). The A element is an 11-bp ARS consensus sequence consisting of (A/T)TTTA(T/C)(A/G)TTT(A/T) that is essential for origin function and ORC binding. Three B elements are located at the 3Ј side of the T-rich strand of the A element. These elements act as auxiliary sequences; they facilitate replication but are dispensable under some conditions. B1 lies adjacent to the A element and facilitates ORC binding. B2 is located further away within an easily unwound DNA sequence referred to as a DNA unwinding element. B3 is found in some, but not all, replication origins, where it is a binding site for transcription factor Abf1. Once a functional ORC:DNA complex is established, it interacts sequentially with Cdc6 and Mcm2-7 proteins to form a prereplication complex. A novel Mcm loading factor, Cdt-1 protein, may also be involved (29). Prereplication complexes are then acted upon by the protein kinases Cdk1/Clb5, Clb6, and Cdc7/Dbf4 to allow Cdc45 to load DNA primase-DNA polymerase ␣ and initiate DNA synthesis. Since homologues of all of these proteins are found throughout the eukaryotic kingdom and since most of them have been shown to be required for initiation of DNA replication in other organisms, the mechanism for initiation of DNA replication appears to be highly conserved among the eukaryotes (5, 22).Two differences between S. cerevisiae and other eukaryotes have emerged that point to the interaction of ORC with DNA replication origins as a critical regulatory step in determi...