Regulation of Interactions with Sliding Clamps During DNA Replication and Repair

The molecular machines that replicate the genome consist of many interacting components. Essential to the organization of the replication machinery are ring-shaped proteins, like PCNA (Proliferating Cell Nuclear Antigen) or the β- clamp, collectively named sliding clamps. They encircle the DNA molecule and slide on it freely and bidirectionally. Sliding clamps are typically associated to DNA polymerases and provide these enzymes with the processivity required to synthesize large chromosomes. Additionally, they interact with a large array of proteins that perform enzymatic reactions on DNA, targeting and orchestrating their functions. In recent years there have been a large number of studies that have analyzed the structural details of how sliding clamps interact with their ligands. However, much remains to be learned in relation to how these interactions are regulated to occur coordinately and sequentially. Since sliding clamps participate in reactions in which many different enzymes bind and then release from the clamp in an orchestrated way, it is critical to analyze how these changes in affinity take place. In this review I focus the attention on the mechanisms by which various types of enzymes interact with sliding clamps and what is known about the regulation of this binding. Especially I describe emerging paradigms on how enzymes switch places on sliding clamps during DNA replication and repair of prokaryotic and eukaryotic genomes

Transposase interaction with the β sliding clamp: Effects on insertion sequence proliferation and transposition rate

Insertion sequences (ISs) are ubiquitous and abundant mobile genetic elements in prokaryotic genomes. ISs often encode only one protein, the transposase, which catalyzes their transposition. Recent studies have shown that transposases of many different IS families interact with the β sliding clamp, a DNA replication factor of the host. However, it was unclear to what extent this interaction limits or favors the ability of ISs to colonize a chromosome from a phylogenetically-distant organism, or if the strength of this interaction affects the transposition rate. Here we describe the proliferation of a member of the IS1634 family in Acidiphilium over ~600 generations of cultured growth. We demonstrate that the purified transposase binds to the β sliding clamp of Acidiphilium, Leptospirillum and E. coli. Further, we also demonstrate that the Acidiphilium IS1634 transposase binds to the archaeal sliding clamp (PCNA) from Methanosarcina, and that the transposase encoded by Methanosarcina IS1634 binds to Acidiphilium β. Finally, we demonstrate that increasing the strength of the interaction between β and transposase results in a higher transposition rate in vivo. Our results suggest that the interaction could determine the potential of ISs to be mobilized in bacterial populations and also their ability to proliferate within chromosomesThis work was funded by the Subdirección General de Proyectos de Investigación of the Spanish Ministry for Economy and Competitiveness Grants No. CGL2010-17384 and AYA2011-24803, and by the European Research Council (ERC) Advanced Grant No. 250350 (IPBSL). HDM has a FPI fellowship from the Spanish Governmen

Quantitative transcription factor binding kinetics at the single-molecule level

We have investigated the binding interaction between the bacteriophage lambda repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large, step-wise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated and dissociated from individually labeled single molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants, ka and kd respectively. We resolved in detail how ka and kd varied as a function of 3 control parameters, the DNA length L, the CI dimer concentration, and the binding affinity. Our results show that although interaction with non-operator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking non-operator DNA.Comment: 34 pages, 10 figures, accepted by Biophysical Journa

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

Chromosomal replication dynamics and interaction with the β sliding clamp determine orientation of bacterial transposable elements

Insertion sequences (ISs) are small transposable elements widespread in bacterial genomes, where they play an essential role in chromosome evolution by stimulating recombination and genetic flow. Despite their ubiquity, it is unclear how ISs interact with the host. Here, we report a survey of the orientation patterns of ISs in bacterial chromosomes with the objective of gaining insight into the interplay between ISs and host chromosomal functions. We find that a significant fraction of IS families present a consistent and family-specific orientation bias with respect to chromosomal DNA replication, especially in Firmicutes. Additionally, we find that the transposases of up to nine different IS families with different transposition pathways interact with the β sliding clamp, an essential replication factor, suggesting that this is a widespread mechanism of interaction with the host. Although we find evidence that the interaction with the β sliding clamp is common to all bacterial phyla, it also could explain the observed strong orientation bias found in Firmicutes, because in this group β is asymmetrically distributed during synthesis of the leading or lagging strands. Besides the interaction with the β sliding clamp, other asymmetries also play a role in the biased orientation of some IS families. The utilization of the highly conserved replication sliding clamps suggests a mechanism for host regulation of IS proliferation and also a universal platform for IS dispersal and transmission within bacterial populations and among phylogenetically distant species

Affinity Purification of an Archaeal DNA Replication Protein Network

Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His6-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His6-tagged proteins, stable complexes assembled in vivo have been isolated directly from clarified cell lysates and the T. kodakarensis proteins present have been identified by mass spectrometry. Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research. The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts

The bright end of the colour-magnitude relation of cluster galaxies

We investigate the development of the red sequence (RS) of cluster galaxies by using a semi-analytic model of galaxy formation. Results show good agreement between the general trend of the simulated RS and the observed relation in early-type galaxies. However, the most luminous galaxies ($M_V \lesssim -20$) depart from the linear fit to observed data, displaying almost constant colours. We analyze the dependence with redshift of the fraction of stellar mass contributed to each galaxy by different processes (i.e., quiescent star formation, disc instability and mergers), finding that the evolution of the bright end, since $z\approx 2$, is mainly driven by minor and major dry mergers. Since the most luminous galaxies have a narrow spread in ages ($1.0\times 10^{10}$ yr $<t<1.2\times 10^{10}$ yr), their metallicities are the main factor that affects their colours. Galaxies in the bright end reach an upper limit in metallicity as a result of the competition of the mass of stars and metals provided by the star formation within the galaxies and by the accretion of merging satellites. Star formation activity in massive galaxies (M_\star \gtrsim 10^{10} M_{\odot}$) contribute with stellar components of high metallicity, but this fraction of stellar mass is negligible. Mergers contribute with a larger fraction of stellar mass ($\approx 10-20$per cent), but the metallicity of the accreted satellites is lower by$\approx 0.2$ dex than the mean metallicity of galaxies they merge with. The effect of dry mergers is to increase the mass of galaxies in the bright end, without significantly altering their metallicities, and hence,their colours, giving rise to the break in the RS. These results are found for clusters with different virial masses, supporting the idea of the universality of the CMR in agreement with observational results.Comment: 18 pages,7 figures,1 table. Accepted for publication in MNRA

The unstructured C-terminus of the τ subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the α subunit

The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τC16. We show that the extreme C-terminal region of τC16 constitutes the site of interaction with α. The τC16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τC16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (KD) of the α−τC16 complex to be ∼260 pM. Competition with immobilized τC16 by τC16 derivatives for binding to α gave values of KD of 7 μM for the α−τC16Δ7 complex. Low-level expression of the genes encoding τC16 and τC16▵7, but not τC16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τC16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τC24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τC16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ

The C-Terminal Domain of the MutL Homolog from Neisseria gonorrhoeae Forms an Inverted Homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage

Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in Bacillus subtilis

In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect. Ribosomal RNA (rRNA) promoters from Gram-positive Bacillus subtilis are regulated by the concentration of their iNTP. Yet, the sequences of these promoters do not emulate the sequence characteristics of [iNTP]-regulated rRNA promoters of Gram-negative Escherichia coli. Here, we identified the 3′-promoter region, corresponding to the transcription bubble, as key for B. subtilis rRNA promoter regulation via the concentration of the iNTP. Within this region, the conserved −5T (3 bp downstream from the −10 hexamer) is required for this regulation. Moreover, we identified a second class of [iNTP]-regulated promoters in B. subtilis where the sequence determinants are not limited to the transcription bubble region. Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis. Finally, this study demonstrates how the same type of regulation can be achieved with strikingly different promoter sequences in phylogenetically distant species