Cdc45-induced loading of human RPA onto single-stranded DNA.

Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8-10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA. This placement reaction required physical contacts of Cdc45 with the RPA70A subdomain. Our results imply that Cdc45 controlled stabilization of the 8-nt RPA binding mode, the subsequent RPA transition into 30-mer mode and facilitated an ordered binding to ssDNA. We propose that a Cdc45-mediated loading guarantees a seamless deposition of RPA on newly emerging ssDNA at the nascent replication fork.

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex.

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical 'wings' of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.

A dual promoter system regulating λ DNA replication initiation.

Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur in vivo and may have important regulatory consequences. This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes.

DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding.

The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.

Coupling of transcription and replication machineries in λ DNA replication initiation: evidence for direct interaction of Escherichia coli RNA polymerase and the λO protein.

Transcription proceeding downstream of the λ phage replication origin was previously shown to support initial steps of the λ primosome assembly in vitro and to regulate frequency and directionality of λ DNA replication in vivo. In this report, the data are presented indicating that the RNA polymerase ß subunit makes a direct contact with the λO protein, a replication initiator of λ phage. These results suggest that the role of RNA polymerase during the initiation of λ phage DNA replication may be more complex than solely influencing DNA topology. Results demonstrated in this study also show that gyrase supercoiling activity stimulates the formation of a complex between λO and RNA polymerase, suggesting that the introduction of negative supercoils by DNA gyrase, besides lowering the energy required for DNA strand separation, may play an additional role in modeling protein-protein interactions at early steps of DNA replication initiation.

Influence of the Escherichia coli oxyR gene function on lambda prophage maintenance.

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of lambda prophage. Here, we demonstrate that H2O2-mediated lambda prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a lambda lysogen in the presence of H2O2. On the other hand, stimulation of the p(M) promoter by cI857 overproduced from a multicopy plasmid was decreased in the DeltaoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the p(M) promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p(M) promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced lambda prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating lambda prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.

The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit plays a role in the CI-dependent activation of the bacteriophage lambda pM promoter.

The bacteriophage lambda p(M) promoter is required for maintenance of the lambda prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the lambda repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of p(M). CI activates p(M) through cooperative binding to the O(R)1 and O(R)2 sites within the O(R) operator, with the O(R)2-bound CI dimer making contact with domain 4 of the RNA polymerase sigma subunit (sigma(4)). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of p(M). We also show that the location of alphaCTD at the p(M) promoter changes in the presence of CI. Thus, in the absence of CI, one alphaCTD is located on the DNA at position -44 relative to the transcription start site, whereas in the presence of CI, alphaCTD is located at position -54, between the CI-binding sites at O(R)1 and O(R)2. These results suggest that contacts between CI and both alphaCTD and sigma are required for efficient CI-dependent activation of p(M).

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci.

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca(2+), a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.

Modulation of lambda plasmid and phage DNA replication by Escherichia coli SeqA protein.

SeqA protein, a main negative regulator of the replication initiation of the Escherichia coli chromosome, also has several other functions which are still poorly understood. It was demonstrated previously that in seqA mutants the copy number of another replicon, the lambda plasmid, is decreased, and that the activity of the lambda p(R) promoter (whose function is required for stimulation of ori lambda) is lower than that in the wild-type host. Here, SeqA-mediated regulation of lambda phage and plasmid replicons was investigated in more detail. No significant influence of SeqA on ori lambda-dependent DNA replication in vitro was observed, indicating that a direct regulation of lambda DNA replication by this protein is unlikely. On the other hand, density-shift experiments, in which the fate of labelled lambda DNA was monitored after phage infection of host cells, strongly suggested the early appearance of sigma replication intermediates and preferential rolling-circle replication of phage DNA in seqA mutants. The directionality of lambda plasmid replication in such mutants was, however, only slightly affected. The stability of the heritable lambda replication complex was decreased in the seqA mutant relative to the wild-type host, but a stable fraction of the lambda O protein was easily detectable, indicating that such a heritable complex can function in the mutant. To investigate the influence of seqA gene function on heritable complex- and transcription-dependent lambda DNA replication, the efficiency of lambda plasmid replication in amino acid-starved relA seqA mutants was measured. Under these conditions, seqA dysfunction resulted in impairment of lambda plasmid replication. These results indicate that unlike oriC, SeqA modulates lambda DNA replication indirectly, most probably by influencing the stability of the lambda replication complex and the transcriptional activation of ori lambda.