ComC is required for the processing and translocation of ComGC, a pilin-like competence protein of Bacillus subtilis

ComGC is a cell surface-localized protein required for DNA binding during transformation in Bacillus subtilis. It resembles type IV prepilins in its N-terminal domain, particularly in the amino acid sequence surrounding the processing cleavage sites of these proteins. ComC is another protein required for DNA binding, which resembles the processing proteases that cleave type IV prepilins. We show here that ComGC is processed in competent cells and that this processing requires ComC. We also demonstrate that the PilD protein of Neisseria gonorrhoeae, a ComC homologue, can process ComGC in Escherichia coli, and that the ComC protein itself is the only B. subtilis protein needed to accomplish cleavage of ComGC in the latter organism. Based on NaOH-solubility studies, we have shown that in the absence of ComC, but in the presence of all other competence proteins, B. subtilis is incapable of correctly translocating ComGC to the outer face of the cell membrane. Finally, we show that ComGC can be cross-linked to yield a form with higher molecular mass, possibly a dimer, and present evidence suggesting that formation of the higher mass complex takes place in the membrane, prior to translocation. Formation of this complex does not require ComC or any of the comG products, other than ComGC itself.     

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.     

Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage

Human (h)PTIP plays important but poorly understood roles in cellular responses to DNA damage. hPTIP interacts with 53BP1 tumour suppressor but only when 53BP1 is phosphorylated by ATM after DNA damage although the mechanism(s) and significance of the interaction of these two proteins are unclear. Here, we pinpoint a single ATM-phosphorylated residue in 53BP1—Ser25—that is required for binding of 53BP1 to hPTIP. Binding of phospho-Ser25 to hPTIP in vitro and in vivo requires two closely apposed pairs of BRCT domains at the C-terminus of hPTIP and neither pair alone can bind to phospho-Ser25, even though one of these BRCT pairs in isolation can bind to other ATM-phosphorylated epitopes. Mutations in 53BP1 and in hPTIP that prevent the interaction of the two proteins, render cells hypersensitive to DNA damage and weaken ATM signalling. The C-terminal BRCT domains of hPTIP are also required for stable retention of hPTIP at sites of DNA damage but this appears to be independent of binding to 53BP1. Thus, the BRCT domains of hPTIP play important roles in the cellular response to DNA damage.     

Structure-Guided Mutational Analysis of a Yeast DEAD-Box Protein Involved in Mitochondrial RNA Splicing

DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context of current models of Mss116p-facilitated splicing.     

Functional residues on the surface of the N-terminal domain of yeast Pms1

Saccharomyces cerevisiae MutLα is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5 Å crystal structure of yeast Pms1 (yPms1) NTD. The structure is similar to the homologous NTDs of Escherichia coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins.     

Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication

During late mitosis and early G₁, a series of proteins are assembled onto replication origins, resulting in them becoming ‘licensed’ for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.     

Sequence-specific interactions of Rep proteins with ssDNA in the AT-rich region of the plasmid replication origin

The DNA unwinding element (DUE) is a sequence rich in adenine and thymine residues present within the origin region of both prokaryotic and eukaryotic replicons. Recently, it has been shown that this is the site where bacterial DnaA proteins, the chromosomal replication initiators, form a specific nucleoprotein filament. DnaA proteins contain a DNA binding domain (DBD) and belong to the family of origin binding proteins (OBPs). To date there has been no data on whether OBPs structurally different from DnaA can form nucleoprotein complexes within the DUE. In this work we demonstrate that plasmid Rep proteins, composed of two Winged Helix domains, distinct from the DBD, specifically bind to one of the strands of ssDNA within the DUE. We observed nucleoprotein complexes formed by these Rep proteins, involving both dsDNA containing the Rep-binding sites (iterons) and the strand-specific ssDNA of the DUE. Formation of these complexes required the presence of all repeated sequence elements located within the DUE. Any changes in these repeated sequences resulted in the disturbance in Rep-ssDNA DUE complex formation and the lack of origin replication activity in vivo or in vitro.     

Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

Rolling circle amplification (RCA) of plasmid or genomic DNA using random hexamers and bacteriophage phi29 DNA polymerase has become increasingly popular in the amplification of template DNA in DNA sequencing. We have found that the mutant protein of single-stranded DNA binding protein (SSB) from Thermus thermophilus (Tth) HB8 enhances the efficiency of amplification of DNA templates. In addition, the TthSSB mutant protein increased the specificity of phi29 DNA polymerase. We have overexpressed the native and mutant forms of TthSSB protein in Escherichia coli and purified them to homogeneity. In vitro, these proteins were found to bind specifically to single-stranded DNA. Addition of TthSSB mutant protein to RCA halved the elongation time required for phi29 DNA polymerase to synthesize DNA fragments in RCA. Furthermore, the presence of the TthSSB mutant protein essentially eliminates nonspecific DNA products in RCA reactions.     

The neutrophil-activating Dps protein of Helicobacter pylori, HP-NAP, adopts a mechanism different from Escherichia coli Dps to bind and condense DNA

The Helicobacter pylori neutrophil-activating protein (HP-NAP), a member of the Dps family, is a fundamental virulence factor involved in H.pylori-associated disease. Dps proteins protect bacterial DNA from oxidizing radicals generated by the Fenton reaction and also from various other damaging agents. DNA protection has a chemical component based on the highly conserved ferroxidase activity of Dps proteins, and a physical one based on the capacity of those Dps proteins that contain a positively charged N-terminus to bind and condense DNA. HP-NAP does not possess a positively charged N-terminus but, unlike the other members of the family, is characterized by a positively charged protein surface. To establish whether this distinctive property could be exploited to bind DNA, gel shift, fluorescence quenching and atomic force microscopy (AFM) experiments were performed over the pH range 6.5–8.5. HP-NAP does not self-aggregate in contrast to Escherichia coli Dps, but is able to bind and even condense DNA at slightly acid pH values. The DNA condensation capacity acts in concert with the ferritin-like activity and could be used to advantage by H.pylori to survive during host-infection and other stress challenges. A model for DNA binding/condensation is proposed that accounts for all the experimental observations.     

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence.

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.     

Dissecting the role of the ?29 terminal protein DNA binding residues in viral DNA replication

Phage ϕ29 DNA replication takes place by a protein-priming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the ϕ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the K_m for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the ϕ29 double-stranded DNA binding protein p6 in the reaction, which decreased the K_m of the DNA polymerase for dATP about 130–190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification.     

Novel histone-like DNA-binding proteins in the nucleoid from the acidothermophillic archaebacterium Sulfolobus acidocaldarius that protect DNA against thermal denaturation

DNA of acidothermophilic archaebacterium Sulfolobus acidocaldarius has a base composition of about 40 mol% G + C content. A low intracellular salt concentration has been inferred for this organism. These features and the high optimal temperature of growth (75°C) would have a destabilising effect on the helical structure of the intracellular DNA. Hence, the nucleoid of this organism has been isolated in order to analyse its proteins composition and to identify any protein factors responsible for stabilisation of the organism's DNA at its growth temperature. The acid-soluble fraction of the nucleoid contains four low-molecular-weight basic proteins. The four proteins have been purified to homogeneity and antibodies to these proteins have been raised in rabbits. Immunodiffusion results suggest that the proteins are antigenically distinct. Three proteins (A, C and C′) stabilise different double-stranded DNA during thermal denaturation and increase T_m of DNA by about 25 C°. These proteins are referred to as helix-stabilising nucleoid proteins (HSNP). Protein B (referred to a DNA-binding nucleoid protein, DBNP-B) does not show helix-stabilising effect. None of the four proteins stabilises double-stranded RNA. The four proteins bind to native and denatured DNA to different extents as measured by DNA-cellulose chromatography and [³H]DNA binding by filtration. We suggest, based on the DNA binding, histone-like and helix-stabilising properties, that the intracellular function of these proteins is to prevent strand separation of DNA at the optimal temperature of growth (75°C).     

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.