Cdc6 and DNA replication: Limited to humble origins

The budding yeast Cdc6 protein is important for regulating DNA replication intiation. Cdc6p acts at replication origins, and cdc6-1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc 18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper⁽¹⁾ further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G₁, and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.     

Cdc6 ATPase activity regulates ORC x Cdc6 stability and the selection of specific DNA sequences as origins of DNA replication

DNA replication, as with all macromolecular synthesis steps, is controlled in part at the level of initiation. Although the origin recognition complex (ORC) binds to origins of DNA replication, it does not solely determine their location. To initiate DNA replication ORC requires Cdc6 to target initiation to specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped mini-chromosome maintenance (MCM) 2-7 DNA helicase component onto DNA. ORC and Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. ORC and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants have reduced activity in ORC x Cdc6 x DNA complex formation. Here we analyzed the role of the Cdc6 ATPase on ORC x Cdc6 complex stability in the presence or absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates ORC x Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the conserved origin A element, and to a lesser extent mutations in the B1 and B2 elements, induce Cdc6 ATPase activity and prevent stable ORC x Cdc6 formation. By analyzing ORC x Cdc6 complex stability on various DNAs, we demonstrated that specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls the rate of Cdc6 dissociation from the ORC x Cdc6 x DNA complex. We propose a mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in a stable ORC x Cdc6 x DNA complex, which can then promote MCM loading. This model has relevance for origin specificity in higher eukaryotes.     

ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae.

We have developed a genomic footprinting protocol which allows us to examine protein-DNA interactions at single copy chromosomal origins of DNA replication in the budding yeast Saccharomyces cerevisiae. We show that active replication origins oscillate between two chromatin states during the cell cycle: an origin recognition complex (ORC)-dependent post-replicative state and a Cdc6p-dependent pre-replicative state. Furthermore, we show that both post- and pre-replicative complexes can form efficiently on closely apposed replicators. Surprisingly, ARS301 which is active as an origin on plasmids but not in its normal chromosomal location, forms ORC- and Cdc6p-dependent complexes in both its active and inactive contexts. Thus, although ORC and Cdc6p are essential for initiation, their binding is not sufficient to dictate origin use.     

The papillomavirus E8-E2C protein represses DNA replication from extrachromosomal origins

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8-E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8-E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8-E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8-E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8-E2C (1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8-E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.     

Molecular evolution of Drosophila Cdc6, an essential DNA replication-licensing gene, suggests an adaptive choice of replication origins

Increased size of eukaryotic genomes necessitated the use of multiple origins of DNA replication, and presumably selected for their efficient spacing to ensure rapid DNA replication. The sequence of these origins remains undetermined in metazoan genomes, leaving important questions about the selective constraints acting on replication origins unanswered. We have chosen to study the evolution of proteins that recognize and define these origins every cell cycle, as a surrogate to the direct analysis of replication origins. Among these DNA replication proteins is the essential Cdc6 protein, which acts to license origins for replication. We find that two different species pairs of Drosophila show evidence of positive selection in Cdc6 in their highly conserved C-terminal AAA-ATPase domain. We also identified amino acid segments that are highly conserved in the N-terminal tail of Cdc6 proteins from various Drosophila species, but are not conserved even in closely related insect species. Instead, we find that the N-terminal tails of Cdc6 proteins vary extensively in size and sequence across different eukaryotic lineages. Our results suggest that choice of origin firing may be significantly altered in closely related species, as each set of replication proteins optimizes to its own genomic landscape.     

Replication fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells

The spatial organization of replicons into clusters is believed to be of critical importance for genome duplication in higher eukaryotes, but its functional organization still remains to be fully clarified. The coordinated activation of origins is insufficient on its own to account for a timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Mechanisms coordinating origin distribution with fork progression are still poorly elucidated, because of technical difficulties of visualizing the process. Taking advantage of a single molecule approach, we delineated and compared the DNA replication kinetics at the genome level in human normal primary and malignant cells. Our results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances. We also found that forks that emanated from closely spaced origins tended to move slower than those associated with long replicons. Taken together, our results indicate a functional role for origin clustering in the dynamic regulation of genome duplication.     

Stable association of mitotic cyclin B/Cdc2 to replication origins prevents endoreduplication

We show that in fission yeast the mitotic B type cyclin Cdc13/Cdc2 kinase associates with replication origins in vivo. This association is dependent on the origin recognition complex (ORC), is established as chromosomes are replicated, and is maintained during G2 and early mitosis. Cells expressing an orp2 (ORC2) allele that reduces binding of Cdc13 to replication origins are acutely prone to chromosomal reduplication. In synchronized endoreduplicating cells, following Cdc13 ablation, replication origins are coordinately licensed prior to each successive round of S phase with the same periodicity as in a normal cell cycle. Thus, ORC bound mitotic Cyclin B/Cdc2 kinase imposes the dependency of S phase on an intervening mitosis but not the temporal licensing of replication origins between each S phase.     

Xenopus Cdc6 performs separate functions in initiating DNA replication

Cdc6 performs an essential role in the initiation of eukaryotic DNA replication by recruiting the minichromosome maintenance (MCM) complex onto DNA. Using immunodepletion/add-back experiments in Xenopus egg extracts, we have determined that both Walker A (ATP binding) and Walker B (ATP hydrolysis) motifs of Xenopus Cdc6 (Xcdc6) are essential, but have distinct functional roles. Although Walker B mutant protein binds chromatin well, Walker A mutant protein binds chromatin poorly. Neither Walker A nor Walker B mutant protein, however, load appreciable MCM onto DNA. Herein, we provide evidence that Cdc6 functions as a multimer: 1) mutant and wild-type Xcdc6 form multimers; 2) either mutant protein is dominant negative when added before wild-type Xcdc6, but stimulates DNA replication when added simultaneously with wild-type Xcdc6; and 3) the two mutants restore DNA replication when added together, in the absence of wild-type Xcdc6. Our findings suggest that ATP may play a key regulatory role within this multimer: its binding to Cdc6 promotes chromatin association and its hydrolysis facilitates MCM loading. Moreover, ATP binding and hydrolysis may occur in trans between Cdc6 subunits within the complex.     

The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1] [2]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3] [4] [5] [6] [7]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) [8] [9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9] [10] [11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.     

Mitochondrial replication origin stability and propensity of adjacent tRNA genes to form putative replication origins increase developmental stability in lizards

Secondary structure stability of mitochondrial origins of light-strand replication (OL) presumably reduces delayed formation of light-strand initiating replication forks on the heavy strand. Delayed replication initiation prolongs single strandedness of the heavy strand. More mutations accumulate during the prolonged time spent single stranded. Presumably, delayed replication initiation and excess mutations affect mitochondrial biochemical processes and ultimately morphological outcomes of development at the whole-organism level. This predicts that developmental stability increases with OL secondary structure stability and with formation of OL-like structures by the five tRNA genes flanking recognized OLs. Stable OLs and high percentages of OL-resembling secondary structures of adjacent tRNA genes (predicted by Mfold) correlate positively with developmental stability in three lizard families (Anguidae, Amphisbaenidae, and Polychrotidae). Accounting for effects of the regular OL, Sfold-predicted OL-like propensity of the entire tRNA gene cluster (not of individual genes) correlates with increased developmental stability in Anguidae, also across the entire free-energy range of Boltzmann's distribution of secondary structures. In the fossorial Amphisbaenidae, the OL-like structure-forming propensity of tRNA genes correlates positively with developmental stability for the distribution's sub-optimally stable regions, and negatively for its optimally stable regions, suggesting the thermoregulated functioning of OL vs. flanking tRNA genes as replication origins. Results for polychrotid tRNA genes are intermediate. Anguid tRNA genes possibly function in addition to the regular OL. Mitochondrial tRNA genes may thus frequently acquire and lose the alternative OL function, without sequence (gene) duplication and loss of their primary function.