Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication.

Geminiviruses are plant DNA viruses with small genomes whose replication, except for the viral replication protein (Rep), depends on host proteins and, in this respect, are analogous to animal DNA tumor viruses, e.g. SV40. The mechanism by which these animal viruses create a cellular environment permissive for viral DNA replication involves the binding of a virally encoded oncoprotein, through its LXCXE motif, to the retinoblastoma protein (Rb). We have identified such a LXCXE motif in the Rep protein of wheat dwarf geminivirus (WDV) and we show its functional importance during viral DNA replication. Using a yeast two-hybrid system we have demonstrated that WDV Rep forms stable complexes with p130Rbr2, a member of the Rb family of proteins, and single amino acid changes within the LXCXE motif abolish the ability of WDV Rep to bind to p130Rbr2. The LXCXE motif is conserved in other members of the same geminivirus subgroup. The presence of an intact Rb binding motif is required for efficient WDV DNA replication in cultured wheat cells, strongly suggesting that one of the functions of WDV Rep may be the linking between viral and cellular DNA replication cycles. Our results point to the existence of a Rb-like protein(s) in plant cells playing regulatory roles during the cell cycle.     

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.     

Plant cells contain a novel member of the retinoblastoma family of growth regulatory proteins.

The product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. Animal virus oncoproteins interact with the Rb protein via an LXCXE motif and disrupt Rb-E2F complexes, driving cells into S-phase. Recently, we found that the RepA protein of a plant geminivirus contains an LXCXE motif that is essential for its function, a finding that predicts the existence of Rb-related proteins in plant cells. Here we report the isolation of a maize cDNA clone encoding a protein (ZmRb1) which, based on structural and functional studies, is closely related to the mammalian Rb family of growth regulatory proteins. ZmRb1 shows a high degree of amino acid conservation when compared with animal Rb members, particularly in the A/B 'pocket' domain, but ZmRb1 has a shorter N-terminal domain. ZmRb1 forms stable complexes with plant LXCXE-containing proteins, e.g. geminivirus RepA protein. Geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding either ZmRb1 or human p130, a member of the Rb family. This suggests that ZmRb1 controls the G1/S transit in plant cells and is consistent with the fact that geminiviruses need an S-phase environment for DNA replication, as animal DNA tumor viruses do. Our results allow the extension of the Rb family of tumor suppressor proteins to plants and have implications on animal and plant strategies for cell growth control.     

植物NAC转录因子的结构功能及其表达调控研究进展

NAC转录因子是近十年来新发现的具有多种生物功能的植物特异转录因子。该家族转录因子的共同特点是其N端为保守的大约150个氨基酸的NAC结构域,C端为高度变异的转录调控区。它们在植物生长发育、激素调节和抵抗逆境等方面发挥着重要的作用。本文主要就植物NAC转录因子的基本结构特征、生物学功能、表达调控及其最新研究进展进行综述。     

Determination of the origin cleavage and joining domain of geminivirus Rep proteins.

Replication of the single-stranded DNA genome of plant geminiviruses follows a rolling circle mechanism. It strictly depends on a 'rolling circle replication initiator protein', the M(r) 41 kDa viral Rep protein, encoded by the C1 or AC1 genes. Using wheat dwarf virus (WDV) and tomato yellow leaf curl virus (TYLCV) as examples, we show that not only the full-size Rep proteins, but also a putative 30 kDa translation product of WDV open reading frame C1-N as well as an artificially shortened 24 kDa Rep of TYLCV, cleave and join single-stranded origin DNA in vitro. Thus the pivotal origin recognition and processing activities of geminivirus Rep proteins must be mediated by the amino-terminal domain of Rep.     

Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis

Using yeast forward and reverse two-hybrid analysis and biochemical techniques, we present novel and definitive in vivo and in vitro evidence that both the N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator protein of Escherichia coli interact physically with plasmid-encoded RepA initiator of pSC101. The N-terminal, but not the C-terminal, region of RepA interacted with DnaA in vitro. These protein-protein interactions are critical for two very early steps of replication initiation, namely origin unwinding and helicase loading. Neither domain I nor IV of DnaA could individually collaborate with RepA to promote pSC101 replication. However, when the two domains are co-expressed within a common cell milieu and allowed to associate non-covalently with each other via a pair of leucine zippers, replication of the plasmid was supported in vivo. Thus, the result shows that physical tethering, either non-covalent or covalent, of domain I and IV of DnaA and interaction of both domains with RepA, are critical for replication initiation. The results also provide the molecular basis for a novel, potential, replication-based bacterial two-hybrid system.     

Substrate recognition by the ClpA chaperone component of ClpAP protease

ClpA, a member of the Clp/Hsp100 ATPase family, is a molecular chaperone and regulatory component of ClpAP protease. We explored the mechanism of protein recognition by ClpA using a high affinity substrate, RepA, which is activated for DNA binding by ClpA and degraded by ClpAP. By characterizing RepA derivatives with N- or C-terminal deletions, we found that the N-terminal portion of RepA is required for recognition. More precisely, RepA derivatives lacking the N-terminal 5 or 10 amino acids are degraded by ClpAP at a rate similar to full-length RepA, whereas RepA derivatives lacking 15 or 20 amino acids are degraded much more slowly. Thus, ClpA recognizes an N-terminal signal in RepA beginning in the vicinity of amino acids 10-15. Moreover, peptides corresponding to RepA amino acids 4-13 and 1-15 inhibit interactions between ClpA and RepA. We constructed fusions of RepA and green fluorescent protein, a protein not recognized by ClpA, and found that the N-terminal 15 amino acids of RepA are sufficient to target the fusion protein for degradation by ClpAP. However, fusion proteins containing 46 or 70 N-terminal amino acids of RepA are degraded more efficiently in vitro and are noticeably stabilized in vivo in clpADelta and clpPDelta strains compared with wild type.     

RINT-1, a novel Rad50-interacting protein, participates in radiation-induced G(2)/M checkpoint control

Rad50, an structural maintenance of chromosomes (SMC) protein family member, participates in a variety of cellular processes, including DNA double-strand break repair, cell cycle checkpoint activation, telomere maintenance, and meiosis. Disruption of Rad50 in mice leads to lethality during early embryogenesis, indicating its essential function in normal proliferating cells. In addition to its ability to form a complex with the DNA double-strand break repair proteins Mre11 and NBS1, Rad50 may interact with other cellular proteins to execute its full range of biological activities. A novel 87-kDa protein named RINT-1 was identified using the C-terminal region of human Rad50 as the bait in a yeast two-hybrid screen. Human RINT-1 shares sequence homology with a novel protein identified in Drosophila melanogaster, including a coiled-coil domain within its N-terminal 150 amino acids, a conserved central domain of about 350 amino acids, and a C-terminal region of 90 amino acids exhibiting 35--38% identity. The conserved central and C-terminal regions of RINT-1 are required for its interaction with Rad50. While Rad50 and RINT-1 are both expressed throughout the cell cycle, RINT-1 specifically binds to Rad50 only during late S and G(2)/M phases, suggesting that RINT-1 may be involved in cell cycle regulation. Consistent with this possibility, MCF-7 cells expressing an N-terminally truncated RINT-1 protein displayed a defective radiation-induced G(2)/M checkpoint. These results suggest that RINT-1 may play a role in the regulation of cell cycle control after DNA damage.     

Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.     

Function of the N-terminal half of RepA in activation of Rts1 ori.

The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37 degrees C. At 42 degrees C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.     

The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo

Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease. To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease. We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells. A mutation in the Walker A box in the C-terminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease. Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site. Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP. These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP. Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair. Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype.     

Characterization of the DNA binding and structural properties of the BRCT region of human replication factor C p140 subunit

BRCT domains, present in a large number of proteins that are involved in cell cycle regulation and/or DNA replication or repair, are primarily thought to be involved in protein-protein interactions. The large (p140) subunit of replication factor C contains a sequence of approximately 100 amino acids in the N-terminal region that binds DNA and is distantly related to known BRCT domains. Here we show that residues 375-480, which include 28 amino acids N-terminal to the BRCT domain, are required for 5'-phosphorylated double-stranded DNA binding. NMR chemical shift analysis indicated that the N-terminal extension includes an alpha-helix and confirmed the presence of a conserved BRCT domain. Sequence alignment of the BRCT region in the p140 subunit of replication factor C from various eukaryotes has identified very few absolutely conserved amino acid residues within the core BRCT domain, whereas none were found in sequences immediately N-terminal to the BRCT domain. However, mapping of the limited number of conserved, surface-exposed residues that were found onto a homology model of the BRCT domain, revealed a clustering on one side of the molecular surface. The cluster, as well as a number of amino acids in the N-terminal alpha-helix, were mutagenized to determine the importance for DNA binding. To ensure minimal structural changes because of the introduced mutations, proteins were checked using one-dimensional (1)H NMR and CD spectroscopy. Mutation of weakly conserved residues on one face of the N-terminal alpha-helix and of residues within the cluster disrupted DNA binding, suggesting a likely binding interface on the protein.     

Molecular Evolution of Drosophila cdc6, an Essential DNA Replication-Licensing Gene,Suggests an Adaptive Choice of Replication Origins

Abstract Increased size of eukaryotic genomes necessitated the use of multiple origins of DNA replication, and presumably selected for their efficient spacing to ensure rapid DNA replication. The sequence of these origins remains undetermined in metazoan genomes, leaving important questions about the selective constraints acting on replication origins unanswered. We have chosen to study the evolution of proteins that recognize and define these origins every cell cycle, as a surrogate to the direct analysis of replication origins. Among these DNA replication proteins is the essential Cdc6 protein, which acts to license origins for replication. We find that two different species pairs of Drosophila show evidence of positive selection in Cdc6 in their highly conserved C-terminal AAA-ATPase domain. We also identified amino acid segments that are highly conserved in the N-terminal tail of Cdc6 proteins from various Drosophila species, but are not conserved even in closely related insect species. Instead, we find that the N-terminal tails of Cdc6 proteins vary extensively in size and sequence across different eukaryotic lineages. Our results suggest that choice of origin firing may be significantly altered in closely related species, as each set of replication proteins optimizes to its own genomic landscape.