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Control of the G1/S transition: the E2F/RBR pathway
As previously described, CYCD/CDKA complexes the first CDK/Cyclin complexes activated for cell cycle onset. Consistently, expression of a number of CYCD responds to external cues (see below). In all eukaryotes, CYCD/CDKA complexes promote the G1/S transiti on by phosphorylating the Retinoblastoma (Rb) protein and alleviating its inhibitory action on E2F transcription factors that can in turn activate genes involved in DNA replication (Berckmans and De Veylder, 2009) (Figure 2). This pathway is conserved in plants, and the Arabidopsis genome encompasses a single Rb homologue (RBR, RetinoBlastoma Related) and six E2Fs (Lammens et al., 2009).
Plant E2F transcription factors can be divided in two sub-groups: canonical E2Fs (E2Fa, b and c) require a Dimerization Partner (DP) to efficiently bind DNA, whereas atypical E2Fs (E2Fd, e and f) function as monomers. Plant E2Fs also differ by their function in cell cycle regulation, E2Fa and b being activators of the cell cycle whereas E2Fc behaves as a negative regulator (Berckmans and De Veylder, 2009).
Upon RBR release, activating E2Fs stimulate the expression of genes required for DNA re plication, including the ones encoding the pre-replication complex (pre-RC). Assembly of the pre-RC on replication origin and DNA replication licensing are key steps to the regulation on the G1/S transition. ORC (origin replication complex) proteins bind to replication origins and recruits CDC6 and CDT1 that in turn allow binding of MCM proteins that function as helicases to open the replication fork (DePamphilis, 2003). All these factors are conserved in Arabidopsis, and interactions between the various constituents of the pre-RC have been observed in the yeast two-hybrid system (Shultz et al., 2007).
Regulation of G2 and mitosis
Many genes expressed during the G2 and M phases harbour a specific regulatory sequence in their promoter called MSA (mitosis-specific activator) (Ito et al., 1998; Menges et al., 2005) that is recognized by MYB3R transcription factors (Haga et al., 2011). Mutants deficient for MYB3R1 and 4 display a drastically reduced stature due to aberrant cytokinesis activate the expression of G2/M specific genes such as KNOLLE to allow proper cytokinesis (Haga et al., 2011).
In addition to the transcriptional regulation of G2/M gene expression, targeted protein degradation plays a pivotal role for progression through mitosis. The Anaphase Promoting Complex/Cyclosome is a highly conserved E3-ubiquitin ligase that specifically targets cell cycle regulators towards proteolysis (Heyman and De Veylder, 2012), that was named for its role in the degradation of the mitosis inhibitor securin (Vodermaier, 2004). This complex comprises 11 sub-units (APC1-11, (Van Leene et al., 2010)), some of which are constitutively expressed while others accumulate specifically during G2 and M (Heyman and De Veylder, 2012) (Figure 2).
Cell Cycle regulation in response to stress
In addition to the programmed changes in cell proliferation associated with normal plant development, the ability to modulate the cell cycle in response to stress is a key parameter for the ability to cope with changing environmental conditions and to adjust their body plan accordingly. As a general rule, stress induces cell differentiation, possibly to avoid the transmission of induced mutations to the progeny of the cells (Cools and De Veylder, 2009). However, CYCB1;1 has the particularity of being induced by genotoxic stress, and has been proposed to function to block some cells in G2, thereby allowing to preserve some proliferative potential until conditions become favourable again (Cools and De Veylder, 2009). In the root of Arabidopsis, replenishment of the meristem after initial cell death is achieved by stimulating the division of quiescent centre (QC) cells that are probably less vulnerable to stress because of their low division rate: when plants are transferred from a medium containing DNA damaging agents back to normal growth medium, the ERF115 transcription factor that is a positive regulator of QC cell division is activating, thereby allowing the replacement of cells that have undergone programmed cell death (Heyman et al., 2013). Yet another mechanism has been described in rice where the RSS1 protein is required to maintain the proliferative capacity of meristematic cells during salt stress (Ogawa et al., 2011), but this factor is not conserved in eudicots. In parallel, stress also induces premature cell differentiation in growing organs. In leaves, drought activates gibberellin signalling and thus stabilization of DELLA proteins that in turn activate the atypical E2F factor E2Fe thereby stimulating the expression of CCS52A and triggering early endoreduplication (Claeys et al., 2012). High light stress also promotes early cell differentiation by activating the expression of the cell cycle inhibitors SMR5 and SMR7 (Hudik et al., 2014; Yi et al., 2014). Likewise, DNA damage causes early differentiation of root meristematic cells (Cools et al., 2011). The analysis of cell cycle progression in response to stress is still in its infancy, but there is also accumulating evidence that biotic stresses also impinge on cell cycle regulation (Reitz et al., 2015), opening exciting new research prospects.
As described above, initiation of DNA replication is one of the key steps of cell cycle regulation because it commits one cell towards division, but can also be the initial step of a di fferentiation programme. In the next section, I will therefore summarize the key regulatory steps in the control of DNA replication in yeast and animals. Indeed, most of the knowledge on eukaryotic DNA replication has been acquired in yeast and animal cells, and the described mechanisms are generally assumed to function in a similar fashion in plants, even though little biochemical evidence is available to fully support this view.
DNA replication during S-phase results in the duplication of the entire genome, which needs to be faithful to avoid problems in gene expression, chromatid cohesion and maintenance of epigenetic features (Costas et al., 2011). DNA replication is a mechanism that involves three steps: initiation, elongation, and termination: the initial steps of DNA replication are depicted on Figure 3. The sequence of events allowing the initiation of DNA replication is so well described now that it has recently been reconstructed in vitro (Yeeles et al., 2015). In this section and throughout the introduction, we will use the appropriate nomenclature for the different eukaryotic organisms we refer to: yeast protein names are spelled in lowercase letters starting with a capital letter (ex: Dpb2), whereas names used in animals are spelled in capital letters (ex: DPB2). For the sake of clarity, when the same protein has a specific name in one model, the name of its homolog in other organisms will be systematically added in brackets.
Regulation of replication initiation
Genome duplication in dividing plant cells has the same requirements and constraints as in animal cells (Sanchez Mde et al., 2012). Thus, the initiation step is strongly regulated because it must occur once and only once per cell cycle. Replication initiation can be divided in two temporally separated steps: the origins first need to be licensed and subsequently to be activated. Thus, initiation of DNA replication in eukaryotes depends on the assembly of pre-replication complex (pre-RC) on many sites of the genome known as replication origins. Pre-RC formation occurs during the late G1-phase of the cell cycle and is called origin licensing; its further activation to initiate DNA replication marks the onset of S-phase and is known as the origin firing process (Hills and Diffley, 2014).
Organisation and function of the replisome (elongation and termination of replication)
DNA replication is a mechanism that leads to the production of two identical sister chromatids that contain one strand from the parental DNA duplex and one new antiparallel strand. This mechanism is conserved from prokaryotes to eukaryotes and is known as semiconservative DNA replication (Leman and Noguchi, 2013). In all living organisms, the DNA replication machinery is a complex and dynamic structure called the replisome. The eukaryotic replisome comprises 48 polypeptides, many of which are absent from the prokaryotic replisome, which reflects the complexity of eukaryotic replication. Replisome assembly occurs only upon entry into S -phase (Kurth and O’Donnel, 2013), and individual subunits are highly regulated by post-translational modifications in a cell cycle dependent manner (Kurth and O’Donnel, 2013) to achieve faithful duplication of the genome.
Structure and properties of DNA polymerases
Pol ε catalyzes DNA template-dependent DNA synthesis by a phosphoryl transfer reaction involving nucleophilic attack by the 3´ hydroxyl of the primer terminus on the α -phosphate of the incoming deoxynucleoside triphosphate (dNTP). The products of this reaction are pyrophosphate and a DNA chain increased in length by one nucleotide. The catalytic mechanism is conserved among DNA polymerases (Lange et al., 2011).
All DNA polymerases share a common polymerase fold, which has been compared to a human right hand, composed of three subdomains; fingers, palm, and thumb. The palm, a highly conserved fold composed of four antiparallel β strands and two helices. In contrast, the thumb and fingers subdomains exhibit substantially more structural diversity. The fingers undergo a conformational change upon binding the DNA template and the correct incoming nucleotide. This movement allows residues in the fingers subdomain to come in contact with the nucleotide in the nascent base pair. The thumb holds the DNA duplex during replication and contributes to processitivity (Doublié and Zahn, 2014).
DNA-dependent DNA polymerases are classified into six families based on primary amino acid sequence similarity in the enzyme active site: A, B, C, D, C, D, X, and Y (Lange et al., 2011). For instance, Y-family DNA polymerases have significantly smaller finger and thumb domains than those of replicative DNA polymerases and spacious active sites that enable them to bypass bulky DNA adducts (Rayner et al., 2016). The three replicative polymerases are part of B-family.
All B family polymerases are formed of five subdomains; the fingers, thumb, and palm are the core of the polymerase activity, whereas an exonuclease domain and an N-terminal domain have independent roles (Doublié and Zahn, 2014).
Table of contents :
I-Cell cycle regulation in plants
A-Plant CDKs and Cyclins, motors of cell cycle progression with an intriguing diversity
B-Control of the G1/S transition: the E2F/RBR pathway
C-Regulation of G2 and mitosis
D-Cell Cycle regulation in response to stress
A-Regulation of replication initiation
B-Organisation and function of the replisome (elongation and termination of replication)
III-Polymerase epsilon (DNA polymerases)
A-Structure and properties of DNA polymerases
B-Specificities of Pol ε subunits
C- Roles of Pol ε subunits at different steps of DNA replication
IV- Mechanisms involved in the maintenance of genome integrity
B-DDR in Mammals and Plants
C- Specific mechanisms triggered by replicative stress
V-Replicative polymerases in plants
B- Polymerase δ
First article: Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis. Published in Nucleic Acids Research (2016, Epub ahead of print).
Second article: Function of the plant DNA Polymerase epsilon in replicative stress sensing, a genetic analysis
Role of Pol sub-units during the replicative stress response in somatic cells
Role of Pol in an adaptation checkpoint involved in DNA damage tolerance
Roles of Pol sub-units during the pre-meiotic DNA replication and meiosis progression
Function of Pol ε subunits
Pol ε complex and interactions
A link between DNA Damage Response and auxin signaling?