No abstract

Helicases are transferred to replication origins by helicase loading factors. The Escherichia coli DnaC and eukaryotic Cdc6/18 helicase loaders contain ATP sites and are both members of the AAA+ family. One might expect that ATP is required for helicase loading; however, this study on DnaC illustrates that ATP is not actually needed for DnaC to load helicase onto single‐strand DNA (ssDNA). In fact, it seems to be a paradox that after transfer of helicase to DNA, DnaC–ATP inhibits helicase action. In addition, ATP is required for DnaC function at an early step in oriC replication in which ATP stimulates ssDNA binding by DnaC, leading to expansion of the ssDNA bubble at the origin. Two cofactors, ssDNA and DnaB, trigger hydrolysis of ATP, converting DnaC to the ADP form that no longer inhibits DnaB. These observations have led to the idea that DnaC is a ‘dual’ switch protein, where both the ATP and the ADP forms are sequentially required for replication. This dual switching process may underlie the sensitivity of DnaB to even small fluctuations in DnaC levels.

We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least 15 chromatin-associated proteins, including DNA pol ɛ, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase. We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.

This study outlines the events downstream of origin unwinding by DnaA, leading to assembly of two replication forks at the E. coli origin, oriC. We show that two hexamers of DnaB assemble onto the opposing strands of the resulting bubble, expanding it further, yet helicase action is not required. Primase cannot act until the helicases move 65 nucleotides or more. Once primers are formed, two molecules of the large DNA polymerase III holoenzyme machinery assemble into the bubble, forming two replication forks. Primer locations are heterogeneous; some are even outside oriC. This observation generalizes to many systems, prokaryotic and eukaryotic. Heterogeneous initiation sites are likely explained by primase functioning with a moving helicase target.

Mcm4,6,7 is a ring-shaped heterohexamer and the putative eukaryotic replication fork helicase. In this study, we examine the mechanism of Mcm4,6,7. Mcm4,6,7 binds to only one strand of a duplex during unwinding, corresponding to the leading strand of a replication fork. Mcm4,6,7 unwinding stops at a nick in either strand. The Mcm4,6,7 ring also actively translocates along duplex DNA, enabling the protein to drive branch migration of Holliday junctions. The Mcm4,6,7 mechanism is very similar to DnaB, except the proteins translocate with opposite polarity along DNA. Mcm4,6,7 and DnaB have different structural folds and evolved independently; thus, the similarity in mechanism is surprising. We propose a "pump in ring" mechanism for both Mcm4,6,7 and DnaB, wherein a single-stranded DNA pump is situated within the central channel of the ring-shaped helicase, and unwinding is the result of steric exclusion. In this example of convergent evolution, the "pump in ring" mechanism was probably selected by eukaryotic and bacterial replication fork helicases in order to restrict unwinding to replication fork structures, stop unwinding when the replication fork encounters a nick, and actively translocate along duplex DNA to accomplish additional activities such as DNA branch migration.

Clamp loaders are required to load the ring-shaped clamps that tether replicative DNA polymerases onto DNA. Recently solved crystal structures, along with a series of biochemical studies, have provided a detailed understanding of the clamp loading reaction. In particular, studies of the Escherichia coli clamp loader--an AAA+ machine--have provided insights into the architecture of clamp loaders from eukaryotes, bacteriophage T4 and archaea. Other AAA+ proteins are also involved in the initiation of DNA replication, and studies of the E. coli clamp loader indicate mechanisms by which these proteins might function.

Helicase loaders transfer the ring-shaped replicative helicases onto DNA. They assort into two classes: ring breakers, which place stabile hexameric rings on DNA via transient gaps at subunit interfaces; and helicase makers, which assemble hexameric rings around DNA from monomeric helicase units.

ParA is an essential P1 plasmid partition protein. It represses transcription of the par genes (parA and parB) and is also required for a second, as yet undefined step in partition. ParA is a ParB-stimulated ATPase that binds to a specific DNA site in the par promoter region. ParA is an essential component of the P1 plasmid partition system (1). The prophage of bacteriophage P1 exists as an autonomously replicating plasmid, and its copy number is about the same as that of the bacterial chromosome (2). The P1 partition system, Par, directs proper segregation and thus stable maintenance of the plasmid. The mechanism of partition is unknown, but can be thought of as a positioning process that via interaction with the Escherichia coli host ensures proper distribution of the plasmid. Par encodes two essential transacting proteins, ParA and ParB, and contains a cis-acting DNA site called parS (1). In addition, the E. coli integration host factor, IHF, 1 also participates, although it is not absolutely required (3). ParB and IHF bind to parS to form the partition complex (3-5). Assembly of this complex is assumed to be an early step in the partition pathway. Formation of this complex does not require the action of ParA (3-5), and we infer that ParA acts during a later step in the partition process.ParA has at least two roles in partition. First, ParA represses transcription of its own gene and parB from a promoter upstream of parA (6). ParA repressor activity is stimulated by ParB; however, ParB has no effect on par gene expression on its own. A second role for ParA in partition is inferred from genetic data that show a requirement for ParA in partition even when its regulatory role is bypassed (6, 7). Therefore, we consider ParA's repressor activity its "regulatory" function, and this second, as yet undefined role as ParA's "partition" function because we think it reflects a direct role for ParA in the positioning reaction. The latter function requires that ParA interact, directly or indirectly, with the ParB-IHF-parS partition complex.ParA is an ATPase and a site-specific DNA-binding protein (8). The ATPase is stimulated by ParB and nonspecific DNA (8). ParA is one of the better characterized proteins in a superfamily of ATPases defined by a modified Walker type A motif in the protein sequence (9 -11). This superfamily includes other plasmid partition proteins such as F SopA, as well as plasmid and chromosomally encoded proteins from various bacteria species whose functions have not yet been determined (10 -12). Many of the plasmids and bacterial chromosomes also encode a ParB homolog adjacent to the ParA homolog. The similarities of these ParA-like proteins with P1 ParA and F SopA have led to the suggestion that these homologs are also involved in plasmid or chromosome segregation (10, 11).ParA binds to the par promoter region called parOP (8), and this binding is thought to mediate ParA repressor activity in vivo. The recognition site for ParA is likely a large inverted repeat in the par promoter region, since (i) ...