Nature of full-length HMGB1 binding to cisplatin-modified DNA

HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.     

Redox state-dependent interaction of HMGB1 and cisplatin-modified DNA

HMGB1, one of the most abundant nuclear proteins, has a strong binding affinity for cisplatin-modified DNA. It has been proposed that HMGB1 enhances the anticancer efficacy of cisplatin by shielding platinated DNA lesions from repair. Two cysteine residues in HMGB1 domain A form a reversible disulfide bond under mildly oxidizing conditions. The reduced domain A protein binds to a 25-bp DNA probe containing a central 1,2-d(GpG) intrastrand cross-link, the major platinum-DNA adduct, with a 10-fold greater binding affinity than the oxidized domain A. The binding affinities of singly and doubly mutated HMGB1 domain A, respectively deficient in one or both cysteine residues that form the disulfide bond, are unaffected by changes in external redox conditions. The redox-dependent nature of the binding of HMGB1 domain A to cisplatin-modified DNA suggests that formation of the intradomain disulfide bond induces a conformational change that disfavors binding to cisplatin-modified DNA. Hydroxyl radical footprinting analyses of wild-type domain A bound to platinated DNA under different redox conditions revealed identical cleavage patterns, implying that the asymmetric binding mode of the protein across from the platinated lesion is conserved irrespective of the redox state. The results of this study reveal that the cellular redox environment can influence the interaction of HMGB1 with the platinated DNA and suggest that the redox state of the A domain is a potential factor in regulating the role of the protein in modulating the activity of cisplatin as an anticancer drug.     

The S. cerevisiae architectural HMGB protein NHP6A complexed with DNA: DNA and protein conformational changes upon binding

NHP6A is a non-sequence-specific DNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Previously, we have solved the structure of NHP6A in the absence of DNA and modeled its interaction with DNA. Here, we present the refined solution structures of the NHP6A-DNA complex as well as the free 15bp DNA. Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provides insight into the factors that contribute to binding site selection and DNA deformations in the complex. Further insight into the amino acid determinants of DNA binding by HMGB domain proteins is given by a correlation study of NHP6A and 32 other HMGB domains belonging to both the DNA-sequence-specific and non-sequence-specific families of HMGB proteins. The resulting correlations can be rationalized by comparison of solved structures of HMGB proteins.     

Binding of the essential Saccharomyces cerevisiae kinetochore protein Ndc10p to CDEII

Chromosome segregation at mitosis depends critically on the accurate assembly of kinetochores and their stable attachment to microtubules. Analysis of Saccharomyces cerevisiae kinetochores has shown that they are complex structures containing >/=50 protein components. Many of these yeast proteins have orthologs in animal cells, suggesting that key aspects of kinetochore structure have been conserved through evolution, despite the remarkable differences between the 125-base pair centromeres of budding yeast and the Mb centromeres of animal cells. We describe here an analysis of S. cerevisiae Ndc10p, one of the four protein components of the CBF3 complex. CBF3 binds to the CDEIII element of centromeric DNA and initiates kinetochore assembly. Whereas CDEIII binding by Ndc10p requires the other components of CBF3, Ndc10p can bind on its own to CDEII, a region of centromeric DNA with no known binding partners. Ndc10p-CDEII binding involves a dispersed set of sequence-selective and -nonselective contacts over approximately 80 base pairs of DNA, suggesting formation of a multimeric structure. CDEII-like sites, active in Ndc10p binding, are also present along chromosome arms. We propose that a polymeric Ndc10p complex formed on CDEII and CDEIII DNA is the foundation for recruiting microtubule attachment proteins to kinetochores. A similar type of polymeric structure on chromosome arms may mediate other chromosome-spindle interactions.     

Saccharomyces cerevisiae replication protein A binds to single-stranded DNA in multiple salt-dependent modes

We have examined the single-stranded DNA (ssDNA) binding properties of the Saccharomyces cerevisiae replication protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry, and sedimentation equilibrium to determine whether scRPA can bind to ssDNA in multiple binding modes. We measured the occluded site size for scRPA binding poly(dT), as well as the stoichiometry, equilibrium binding constants, and binding enthalpy of scRPA-(dT)L complexes as a function of the oligodeoxynucleotide length, L. Sedimentation equilibrium studies show that scRPA is a stable heterotrimer over the range of [NaCl] examined (0.02-1.5 M). However, the occluded site size, n, undergoes a salt-dependent transition between values of n = 18-20 nucleotides at low [NaCl] and values of n = 26-28 nucleotides at high [NaCl], with a transition midpoint near 0.36 M NaCl (25.0 degrees C, pH 8.1). Measurements of the stoichiometry of scRPA-(dT)L complexes also show a [NaCl]-dependent change in stoichiometry consistent with the observed change in the occluded site size. Measurements of the deltaH(obsd) for scRPA binding to (dT)L at 1.5 M NaCl yield a contact site size of 28 nucleotides, similar to the occluded site size determined at this [NaCl]. Altogether, these data support a model in which scRPA can bind to ssDNA in at least two binding modes, a low site size mode (n = 18 +/- 1 nucleotides), stabilized at low [NaCl], in which only three of its oligonucleotide/oligosaccharide binding folds (OB-folds) are used, and a higher site size mode (n = 27 +/- 1 nucleotides), stabilized at higher [NaCl], which uses four of its OB-folds. No evidence for highly cooperative binding of scRPA to ssDNA was found under any conditions examined. Thus, scRPA shows some behavior similar to that of the E. coli SSB homotetramer, which also shows binding mode transitions, but some significant differences also exist.     

Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA

High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.     

Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a ∼1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21–24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20–50 nucleotides was ∼3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.MCM10 is a ubiquitous, conserved gene essential for DNA replication in eukaryotes. It was first discovered in yeast genetic screens designed to detect mutants defective in DNA synthesis and minichromosome maintenance (1, 2). In vivo, Mcm10 associates with chromatin and chromosomal replication origins in human cells (hMcm10), Xenopus laevis (XMcm10), Schizosaccharomyces pombe (SpMcm10), and Saccharomyces cerevisiae (ScMcm10) (3–6). In S. cerevisiae, initiation of chromosomal replication occurs at multiple specific sites known as autonomously replicating sequences (ARSs)² (7). Mutations in MCM10 enhance the loss rate of plasmids bearing specific ARSs (8), suggesting a function for ScMcm10 in initiation.In eukaryotic systems replication initiation is a cell cycle-regulated process characterized by a multistep sequential loading of ORC, Cdc6, Cdt1, and the Mcm2–7 complex onto the origin in G₁ to form the pre-RC complex. Binding of ORC, Cdc6p, and Cdt1p to chromatin is a prerequisite for the recruitment of Mcm2–7 (9, 10). The next step in the assembly of the initiation replication apparatus in S. cerevisiae involves protein kinases (Cdc28 and Cdc7/Dbf4), and recruitment of Mcm10, Cdc45, and the GINS complex. The mechanism for targeting Mcm10 to replications origins is unknown. However, recent studies in S. cerevisiae have shown that Mcm10 and Mcm2–7 physically interact (6, 11). It is now believed that in late G₁, chromatin-bound Mcm2–7 is responsible for the recruitment of Mcm10 presumably via protein-protein interactions (12). Prior studies in the Xenopus laevis system reached similar conclusions (4). Additional interactions of Mcm10 with other components of the pre-RC cannot be excluded (13).A key step in the initiation of replication is the local melting of an origin DNA sequence, which occurs at the G₁/S transition and throughout the S phase. The mechanism of this essential DNA-melting process is not understood. There is no evidence in S. cerevisiae that the assembled pre-RC complex leads to the melting of an origin DNA sequence. This unwinding may occur only following the recruitment of Mcm10, raising the possibility that Mcm10 is a key component of the initiation machinery responsible for this process. Results of a study in the Xenopus egg extract system (4), which showed that omission of XMcm10 blocks unwinding of plasmid DNA and initiation of DNA replication, are consistent with this notion. An additional function of Mcm10 in initiation is in the recruitment of Cdc45 to replication origins, presumably via Mcm10-Cdc45 physical interactions (5, 14). Cdc45 is believed to be important for the activation of replication origins and the assembly of the replication elongation complex (15). Upon initiation of DNA replication, ScMcm10 moves from the origin to origin-proximal sequences suggesting that ScMcm10 associates with moving replication forks (12) and is consistent with the observation that elevated temperatures cause pausing of replication forks in a mcm10-1 ts mutant (8). Both ScMcm10 and SpMcm10 interact with DNA polymerase α supporting the notion that replication fork movement requires Mcm10. ScMcm10 and polymerase α form a complex on and off the DNA in vivo (12). In S. pombe, SpMcm10 stimulates the activity of polymerase α in vitro and associates with a primase activity (16, 17) that has not been reported in other eukaryotes (18).Previous studies with Mcm10 in other systems showed that Mcm10 binds DNA. Using a filter binding assay Fien and Hurwitz (16) reported that SpMcm10 from S. pombe binds well to ssDNA but barely interacts (20-fold lower affinity) with dsDNA. It has been suggested that binding of SpMcm10 to ssDNA is important for the recruitment of polymerase α (16). Recently, it has been reported that a DNA binding activity is also associated with XMcm10 protein from X. laevis. Measurements of fluorescence anisotropy were used to show binding of XMcm10 to short, 25-nucleotide-long oligonucleotides (18). These studies have shown that XMcm10 has similar affinities for ssDNA and dsDNA. Unlike SpMcm10, which harbors a single DNA-binding domain in the N-terminal half of the protein, XMcm10 seems to contain two distinct domains for binding DNA. The biological implication of having two DNA-binding domains is not clear.It appears that there are differences in the quaternary structure of Mcm10 from different organisms. Although SpMcm10 and XMcm10 may be a homodimer in solution (17, 18), a recent electron microscopy study suggested that human hMcm10 has a hexameric ring structure (19). The same study reported that hMcm10 interacts with ssDNA but failed to bind dsDNA. The differences in structure and DNA binding properties may reflect differences in the function of Mcm10 in various organisms as well as in the protein preparations.Here we report, for the first time, the characterization of the DNA binding properties of purified Mcm10 from S. cerevisiae. We show that ScMcm10 forms a stable complex with dsDNA and ssDNA. In addition, we demonstrate that dsDNA longer than 50 bp sustains oligomerization of ScMcm10. The number of ScMcm10 molecules bound is directly proportional to the size of the dsDNA, suggesting that ScMcm10 is tightly packed on the dsDNA, perhaps in a head to tail oligomeric structure. In contrast to a 25-bp-long dsDNA, which supports the binding of a single copy of ScMcm10, ssDNA containing only 20 nucleotides may sustain binding of as many as three copies of ScMcm10, suggesting that a ScMcm10 complex composed of at least 3 subunits assembles on ssDNA. We believe that these distinct binding properties to dsDNA and ssDNA are important for the ScMcm10 functions in initiation, formation of replication forks, and the maintenance of replication fork progression during chromosomal DNA replication.     

Binding and partial denaturing of G-quartet DNA by Cdc13p of Saccharomyces cerevisiae

The protein Cdc13p binds telomeres in vivo and is essential for the maintenance of the telomeres of Saccharomyces cerevisiae. In addition, Cdc13p is known to bind single-stranded TG(1-3) DNA in vitro. Here we have shown that Cdc13p also binds DNA quadruplex, G-quartet, formed by TG(1-3) DNA. Moreover, the binding of Cdc13p causes a partial denaturing of the G-quartet DNA. Formation of DNA quadruplexes may involve the intermolecular association of TG(1-3) DNA and inhibit the extension of telomeres by telomerase. Thus, our finding suggests that Cdc13p may disrupt telomere association and facilitate telomere replication.     

Binding of Saccharomyces cerevisiae extracellular proteins to glucane.

Interactions of Saccharomyces cerevisiae cell wall proteins with purified yeast glucane were studied. Using the beta-glucanase (BGL2 gene product) as the model cell wall protein, strong binding to glucane was demonstrated at pH lower than 7, while at pH higher than 8 the reaction did not occur. NaCl (2 M) did not influence the binding, while urea in concentrations higher than 4 M affected the interactions. It was also found that most other cell wall proteins, as well as intracellular proteins, reacted with glucane in the same way, showing that the interactions of proteins with glucane are rather nonspecific. Soluble periplasmic proteins invertase and acid phosphatase failed to react with glucane under the same conditions, indicating that these proteins have certain structural features preventing their interactions with glucane.     

Control of ATP-Dependent Binding of Saccharomyces cerevisiae Origin Recognition Complex to Autonomously Replicating DNA Sequences

Eukaryotic origin recognition complexes (ORCs) play pivotal roles in the initiation of chromosomal DNA replication. ORC from the yeast, Saccharomyces cerevisiae, recognizes and binds replication origins in the late G1 phase and the binding has profound implications in the progression of the cell cycle to the S-phase. Therefore, we have quantitatively analyzed the mechanism of recognition and interaction of the yeast ORC with various elements of a yeast origin of DNA replication, the autonomously replicating sequence 1 (ARS1). ORC bound all four individual A and B elements of ARS1 with reasonably high affinities. However, the highest affinity binding was observed with a DNA sequence containing both the A and B1 elements. In addition, ATP and ADP significantly modulated the binding of ORC to the combined elements as well as modulating the binding of ORC to the element A alone or in combination with the B1 element. However, binding of ORC to individual B1, B2, and B3 elements was not responsive to nucleotides. Thus, the consensus ARS sequence in element A appeared to play a pivotal role in the ATP-dependent binding of ORC to ARS1 and likely in other ARSs or origins of DNA replication.     

A DNA integrity network in the yeast Saccharomyces cerevisiae

A network governing DNA integrity was identified in yeast by a global genetic analysis of synthetic fitness or lethality defect (SFL) interactions. Within this network, 16 functional modules or minipathways were defined based on patterns of global SFL interactions. Modules or genes involved in DNA replication, DNA-replication checkpoint (DRC) signaling, and oxidative stress response were identified as the major guardians against lethal spontaneous DNA damage, efficient repair of which requires the functions of the DNA-damage checkpoint signaling and multiple DNA-repair pathways. This genome-wide genetic interaction network also identified novel components (DIA2, NPT1, HST3, HST4, and the CSM1 module) that potentially contribute to mitotic DNA replication and genomic stability and revealed novel functions of well-studied genes (the CTF18 module) in DRC signaling. This network will guide more detailed characterization of mechanisms governing DNA integrity in yeast and other organisms.     

A function for subtelomeric DNA in Saccharomyces cerevisiae

The subtelomeric DNA sequences from chromosome I of Saccharomyces cerevisiae are shown to be inherently poor substrates for meiotic recombination. On the basis of these results and prior observations that crossovers near telomeres do not promote efficient meiosis I segregation, we suggest that subtelomeric sequences evolved to prevent recombination from occurring where it cannot promote efficient segregation.