The bacterial cell cycle: DNA replication, nucleoid segregation, and cell division

Data on the bacterial cell cycle published in the last 10-15 years are considered, with a special stress on studies of nucleoid segregation between dividing cells. The degree of similarity between the eukaryotic mitotic apparatus and the apparatus performing nucleoid separation is discussed.     

Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation

Background

PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome.

Scope

This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution.

Conclusions

Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.     

Mathematical models of cell cycle regulation

The cell division cycle is a fundamental process of cell biology and a detailed understanding of its function, regulation and other underlying mechanisms is critical to many applications in biotechnology and medicine. Since a comprehensive analysis of the molecular mechanisms involved is too complex to be performed intuitively, mathematical and computational modelling techniques are essential. This paper is a review and analysis of recent approaches attempting to model cell cycle regulation by means of protein-protein interaction networks.     

DNA replication and cell cycle in plants: learning from geminiviruses

Plant cell growth and development depend on continuous cell proliferation which is restricted to small regions of the plant called meristems. Infection by geminiviruses, small DNA viruses whose replicative cycle relies on host cell factors, is excluded from those proliferating areas. Since most of the replicative factors are present, almost exclusively, in proliferating cells, geminivirus infection is believed to induce a cellular state permissive for viral DNA replication, e.g. S-phase or, at least, some specific S-phase functions. The molecular basis for this effect seems to be the interference that certain geminivirus proteins exert on the retinoblastoma-related (RBR) pathway, which analogously to that of animal cells, regulates plant cell cycle activation and G(1)-S transition. In some cases, geminiviruses induce cell proliferation and abnormal growth. Mechanisms other than sequestering plant RBR probably contribute to the multiple effects of geminivirus proteins on cellular gene expression, cell growth control and cellular DNA replication. Current efforts to understand the coupling of geminivirus DNA replication to cell cycle and growth control as well as the directions in which future research is aiming are reviewed.     

The nuclear envelope in the plant cell cycle: structure, function and regulation

Background

Higher plants are, like animals, organisms in which successful completion of the cell cycle requires the breakdown and reformation of the nuclear envelope in a highly controlled manner. Interestingly, however, while the structures and processes appear similar, there are remarkable differences in protein composition and function between plants and animals.

Scope

Recent characterization of integral and associated components of the plant nuclear envelope has been instrumental in understanding its functions and behaviour. It is clear that protein interactions at the nuclear envelope are central to many processes in interphase and dividing cells and that the nuclear envelope has a key role in structural and regulatory events.

Conclusion

Dissecting the mechanisms of nuclear envelope breakdown and reformation in plants is necessary before a better understanding of the functions of nuclear envelope components during the cell cycle can be gained.     

Break dosage, cell cycle stage and DNA replication influence DNA double strand break response

DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5' DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3' ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.     

Genetic mouse models to investigate cell cycle regulation

Early studies on cell cycle regulation were based on experiments in model systems (Yeast, Xenopus, Starfish, Drosophila) and have shaped the way we understand many events that control the cell cycle. Although these model systems are of great value, the last decade was highlighted by studies done in human cells and using in vivo mouse models. Mouse models are irreplaceable tools for understanding the genetics, development, and survival strategies of mammals. New developments in generating targeting vectors and mutant mice have improved our approaches to study cell cycle regulation and cancer. Here we summarize the most recent advances of mouse model approaches in dissecting the mechanisms of cell cycle regulation and the relevance to human disease. W. Li and S. Kotoshiba contributed equally.     

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.     

Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle

Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two‐ to four‐cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4‐1/+ with AtRFC4 expression driven through the embryo‐specific DD45pro and ABI3pro or the endosperm‐specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4‐1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4‐1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S‐phase and M‐phase cells in the rfc4‐1/–^{FIS2;DD45;ABI3pro::AtRFC4} seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication.     

Mechanisms restricting DNA replication to once per cell cycle: MCMS, pre-replicative complexes and kinases

An important aspect of cell behaviour is that DNA replication happens only once per cell cycle. Replicated DNA is unable to re-replicate until cell division has occurred. Unreplicated DNA is in a replication-competent or 'licensed' state. The ability to replicate is lost in S phase and regained following passage through mitosis. Recent evidence has implicated an MCM (minichromosome maintenance) protein complex and the Cdc6 protein in determining replication competence. Regeneration of replication competence upon passage through mitosis entails changes in protein kinase activity, of which the MCMs are a likely target. Features of the mechanism that restricts DNA replication to once per cell cycle appear to be conserved throughout eukaryotes.