How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

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Mechanisms and regulation of DNA replication initiation in eukaryotes

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms.     

Bacteriophage replication modules

Bacteriophages (prokaryotic viruses) are favourite model systems to study DNA replication in prokaryotes, and provide examples for every theoretically possible replication mechanism. In addition, the elucidation of the intricate interplay of phage-encoded replication factors with 'host' factors has always advanced the understanding of DNA replication in general. Here we review bacteriophage replication based on the long-standing observation that in most known phage genomes the replication genes are arranged as modules. This allows us to discuss established model systems--f1/fd, phiX174, P2, P4, lambda, SPP1, N15, phi29, T7 and T4--along with those numerous phages that have been sequenced but not studied experimentally. The review of bacteriophage replication mechanisms and modules is accompanied by a compendium of replication origins and replication/recombination proteins (available as supplementary material online).     

A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication

In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.     

Structural and functional insights into the DNA replication factor Cdc45 reveal an evolutionary relationship to the DHH family of phosphoesterases

Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication and the progression of the replication fork. With GINS, Cdc45 is an essential cofactor of the Mcm2-7 replicative helicase complex. Despite its importance, no detailed information is available on either the structure or the biochemistry of the protein. Intriguingly, whereas homologues of both GINS and Mcm proteins have been described in Archaea, no counterpart for Cdc45 is known. Herein we report a bioinformatic analysis that shows a weak but significant relationship among eukaryotic Cdc45 proteins and a large family of phosphoesterases that has been described as the DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyze the hydrolysis of phosphodiester bonds via a mechanism involving two Mn(2+) ions. Only a subset of the amino acids that coordinates Mn(2+) is conserved in Cdc45. We report biochemical and structural data on the recombinant human Cdc45 protein, consistent with the proposed DHH family affiliation. Like the RecJ exonucleases, the human Cdc45 protein is able to bind single-stranded, but not double-stranded DNA. Small angle x-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members.     

Dephosphorylation of the pre-initiation complex is critical for origin firing

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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.     

Replication dynamics in fission and budding yeasts through DNA polymerase tracking

The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof‐read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions.     

Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe

DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)‐rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T‐rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25–30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low‐efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals.     

Tel1ATM dictates the replication timing of short yeast telomeres

Telomerase action is temporally linked to DNA replication. Although yeast telomeres are normally late replicating, telomere shortening leads to early firing of subtelomeric DNA replication origins. We show that double‐strand breaks flanked by short telomeric arrays cause origin firing early in S phase at late‐replicating loci and that this effect on origin firing time is dependent on the Tel1^ATM checkpoint kinase. The effect of Tel1^ATM on telomere replication timing extends to endogenous telomeres and is stronger than that elicited by Rif1 loss. These results establish that Tel1^ATM specifies not only the extent but also the timing of telomerase recruitment.     

CDT1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis

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Stalled replication forks: Making ends meet for recognition and stabilization

In bacteria, PriA protein, a conserved DEXH‐type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA‐binding property allows it to recognize and stabilize stalled forks and the structures derived from them. Cells must cope with fork stalls caused by various replication stresses to complete replication of the entire genome. Failure of the stalled fork stabilization process and eventual restart could lead to various forms of genomic instability. The low viability of priA null cells indicates a frequent occurrence of fork stall during normal growth that needs to be properly processed. PriA specifically recognizes the 3′‐terminus of the nascent leading strand or the invading strand in a displacement (D)‐loop by the three‐prime terminus binding pocket (TT‐pocket) present in its unique DNA binding domain. Elucidation of the structural basis for recognition of arrested forks by PriA should provide useful insight into how stalled forks are recognized in eukaryotes.     

Mechanism of Bidirectional Leading-Strand Synthesis Establishment at Eukaryotic DNA Replication Origins

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Establishment of a replication fork barrier following induction of DNA binding in mammalian cells

Understanding the mechanisms that lead to replication fork blocks (RFB) and the means to bypass them is important given the threat that they represent for genome stability if inappropriately handled. Here, to study this issue in mammals, we use integrated arrays of the LacO and/or TetO as a tractable system to follow in time a process in an individual cell and at a single locus. Importantly, we show that induction of the binding by LacI and TetR proteins, and not the presence of the repeats, is key to form the RFB. We find that the binding of the proteins to the arrays during replication causes a prolonged persistence of replication foci at the site. This, in turn, induces a local DNA damage repair (DDR) response, with the recruitment of proteins involved in double-strand break (DSB) repair such as TOPBP1 and 53BP1, and the phosphorylation of H2AX. Furthermore, the appearance of micronuclei and DNA bridges after mitosis is consistent with an incomplete replication. We discuss how the many DNA binding proteins encountered during replication can be dealt with and the consequences of incomplete replication. Future studies exploiting this type of system should help analyze how an RFB, along with bypass mechanisms, are controlled in order to maintain genome integrity.