Interplay between in vitro acetylation and phosphorylation of tailless HMGB1 protein

The postsynthetic acetylation of HMGB1 protein and its truncated form affects significantly its properties as “architectural” factor - recognition of bent DNA and bending of short DNA fragments. We created mutants at the target sites (lysines 2 and 81) in the tailless HMGB1 modified by the histone acetyltransferase CBP. The results show that there is no preferential site for the enzymatic activity of CBP and both lysine moieties are modified independently. Our findings for the first time demonstrate the link between the acetylation and phosphorylation of HMGB1ΔC in vitro. The PKC phosphorylation prior to acetylation inhibits the CBP activity 40-60% for the truncated form and its mutants. The effect of the CBP acetylation on the phosphorylation level turns out to be much more prominent. In the case of HMGB1ΔC modified at Lys 2 and Lys 81 prior to PKC treatment background phosphorylation is detected. If only one of the lysines is modified the inhibitory effect decreases.     

Post-synthetic acetylation of HMGB1 protein modulates its interactions with supercoiled DNA

High mobility group box (HMGB) proteins 1 and 2 are abundant non-histone nuclear proteins that regulate chromatin structure because of their structure-specific binding to DNA. Here, we have investigated how the post-synthetic acetylation of HMGB1 affects its interaction with negatively supercoiled DNA by employing monoacetylated at Lys2 protein, isolated from butyrate-treated cells. Our data reveal that this modification enhances three reaction parameters: binding affinity, supercoiling activity and capacity to protect the supercoiled DNA from relaxation by topoisomerase I. We show that monoacetylation at Lys2 mimics the effect of acidic tail removal but to a lesser extent thus demonstrating that in vivo acetylated HMGB1 is capable of modulating its interaction with negatively supercoiled DNA.     

Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently

We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.     

The DNA binding and bending activities of truncated tail-less HMGB1 protein are differentially affected by Lys-2 and Lys-81 residues and their acetylation

The role of lysines 2 and 81 as target sites for acetylation in full-length HMGB1 and truncated tail-less protein, respectively, has been studied by mutation analysis for the abilities of these proteins to bind and bend DNA. The DNA bending ability of truncated tail-less HMGB1 containing Lys-2 mutated to alanine does not differ from that of the wild-type protein, while the same mutation of Lys-81 reduced the bending capacity of the mutant protein. These data demonstrate that Lys-81 is critical for the DNA bending ability of truncated HMGB1. Such a conclusion is further confirmed by the experiments carried out with CBP-acetylated proteins: acetylation of Lys-2 in mutant protein K81/A81 alleviated DNA bending and induced DNA end-joining. On the contrary, the acetylation of Lys-81 in the mutant K2/A2 enhanced the bending potential of HMGB1∆C. Regarding the ability of HMGB1 to specifically bind bent DNA, the individual mutations of either K2 or K81 as well as the double mutation of both residues to alanine were found to completely abolish binding of truncated tail-less HMGB1 to cisplatin-modified DNA. We conclude that unlike the case with the bending ability of truncated HMGB1, where Lys-81 has a primary function, Lys-2 and Lys-81 are both critical for the protein''s binding to cisplatin-modified DNA. The mutation K2/A2 in full-length HMGB1 and acidic tail removal induce the same conformational changes. Any further substitutions at the acetylable lysines in the truncated form of HMGB1 do not have an additional effect.     

The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1ΔC) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1ΔC were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.     

The role of SMARCAL1 in replication fork stability and telomere maintenance

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1), also known as HARP, is an ATP-dependent annealing helicase that stabilizes replication forks during DNA damage. Mutations in this gene are the cause of Schimke immune-osseous dysplasia (SIOD), an autosomal recessive disorder characterized by T-cell immunodeficiency and growth dysfunctions. In this review, we summarize the main roles of SMARCAL1 in DNA repair, telomere maintenance and replication fork stability in response to DNA replication stress.     

Dynamic elements of replication protein A at the crossroads of DNA replication,recombination, and repair

Abstract

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.     

The interaction of HMGB1 and linker histones occurs through their acidic and basic tails

H1 and HMGB1 bind to linker DNA in chromatin, in the vicinity of the nucleosome dyad. They appear to have opposing effects on the nucleosome, H1 stabilising it by "sealing" two turns of DNA around the octamer, and HMGB1 destabilising it, probably by bending the adjacent DNA. Their presence in chromatin might be mutually exclusive. Displacement/replacement of one by the other as a result of their highly dynamic binding in vivo might, in principle, involve interactions between them. Chemical cross-linking and gel-filtration show that a 1:1 linker histone/HMGB1 complex is formed, which persists at physiological ionic strength, and that complex formation requires the acidic tail of HMGB1. NMR spectroscopy shows that the linker histone binds, predominantly through its basic C-terminal domain, to the acidic tail of HMGB1, thereby disrupting the interaction of the tail with the DNA-binding faces of the HMG boxes. A potential consequence of this interaction is enhanced DNA binding by HMGB1, and concomitantly lowered affinity of H1 for DNA. In a chromatin context, this might facilitate displacement of H1 by HMGB1.     

Interferon‐inducible protein,P56, inhibits HPV DNA replication by binding to the viral protein E1

Type I interferon (IFN) inhibits, by an unknown mechanism, the replication of human papillomaviruses (HPV), which are major human pathogens, Here, we present evidence that P56 (a protein), the expression of which is strongly induced by IFN, double‐stranded RNA and viruses, mediates the anti‐HPV effect of IFN. Ectopic expression of P56 inhibited HPV DNA replication and its ablation in IFN‐treated cells alleviated the inhibitory effect of IFN on HPV DNA replication. Protein–protein interaction and mutational analyses established that the antiviral effect of P56 was mediated by its direct interaction with the DNA replication origin‐binding protein E1 of several strains of HPV, through the tetratricopeptide repeat 2 in the N‐terminal region of P56 and the C‐terminal region of E1. In vivo, the interaction with P56, a cytoplasmic protein, caused translocation of E1 from the nucleus to the cytoplasm. In vitro, recombinant P56, or a small fragment derived from it, inhibited the DNA helicase activity of E1 and E1‐mediated HPV DNA replication. These observations delineate the molecular mechanism of IFN's antiviral action against HPV.     

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.     

Prereplicative complexes assembled in vitro support origin‐dependent and independent DNA replication

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2‐7 helicase is first loaded into prereplicative complexes (pre‐RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre‐RCs assembled with purified proteins support complete and semi‐conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK). DDK phosphorylation of Mcm2‐7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin‐dependent in this system. These experiments indicate that Mcm2‐7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.     

Identification of cellular components required for SV40 DNA replication in vitro

To investigate the cellular proteins involved in simian virus 40 (SV40) replication, extracts derived from human 293 cells have been fractionated into multiple components. When such fractions are combined with the virus-encoded T antigen (TAg) and SV40 origin containing plasmid DNA, efficient and complete replication is achieved, while each fraction alone is inactive. At present, a minimum of eight such cellular components have been identified. Previous experiments have demonstrated one of these to be the cell-cycle-regulated proliferating-cell nuclear antigen (PCNA). As PCNA has been identified as a processivity factor for DNA polymerase δ, we suggest that both polymerases α and β are involved in this system. Three further fractions have been identified. One is a partially purified fraction which, under certain conditons, is required with TAg for the formation of a pre-synthesis complex of proteins at the replication origin. The second of these factors, RF-A, is a complex of three polypeptides which may function as a eucaryotic SSB. The third, RF-C, is a factor which is required, with PCNA, for coordinated leading- and lagging-strand synthesis at the replication fork. Complete synthesis and segregation of the daughter molecules also requires the presence of topoisomerases I and II. These results suggest a model for DNA synthesis which involves multiple stages prior to and during replicative DNA synthesis.     

Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control

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Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication

Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase alpha-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein-protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end.     

Biochemical regulation of the initiation of DNA replication

Abstract

The initiation of DNA replication is a key step in the cell division cycle and in DNA endoreduplication. Initiation of replication takes place at specific places in chromosomes known as replication origins. These are subject to temporal regulation within the cell cycle and may also be regulated as a function of plant development. In yeast, replication origins are recognised and bound by three different groups of proteins at different stages of the cell cycle. Of these, the MCM proteins are the most likely to be involved in activating the origins in order to facilitate initiation. MCM-like proteins also occur in plants, but have not been characterised in detail. Other proteins which bind to origins have been identified, as has a protein with a strong affinity for ds-ss junctions in DNA molecules.     

Cellular localisation of the clamp protein during DNA replication

The beta subunit of Escherichia coli DNA polymerase III holoenzyme was fused to the green fluorescent protein GFP. The gene fusion under the control of the heterologous lac promoter was used to replace the wild-type allele in the chromosome. The formation of GFP-beta fluorescent foci in GFP-beta expressing cells required DNA replication and their number per cell was dependent on cell growth. Examination of GFP-beta foci in a synchronous round of replication suggested that DNA replication was accompanied by the recruitment of GFP-beta foci near the midcell, followed by the rapid migration of the foci in opposite directions to the 1/4 and 3/4 positions during DNA replication.