In eukaryotes, initiation of DNA replication requires the assembly of pre-replication complexes (pre-RCs) on chromatin during the G1 phase. In the S phase, pre-RCs are activated by two protein kinases, Cdk2 and Cdc7, which results in the loading of replication factors and the unwinding of replication origins by the MCM helicase complex []. Cdc7 is a serine/threonine kinase that is conserved from yeast to human. It is regulated by its association with a regulatory subunit, the Dbf4 protein. This complex is often referred to as DDK (Dbf4-dependent kinase) [].DBF4 contains an N-terminal BRCT domain and a C-terminal conserved region that could potentially coordinate one zinc atom, the DBF4-type zinc finger. This entry represents the zinc finger, which is important for the interaction with Cdc7 [, ].
Cyclin-dependent kinase 2-interacting protein (CINP) is a component of theactive cyclin E/Cdk2 and cyclin A/Cdk2 complexes []. It is phopshorylated by Cdc7, but not by Cdk2 []. CINP has also been shown to bind to chromatin in a replication-dependent manner, and to associate with Origin Recognition Complex-2 (ORC2)-containing complexes and minichromosome maintenance/DNA replication licensing complex, MCM []. It has been proposed that CINP is part of the Cdc7-dependent mechanism of origin firing, and constitutes a physical link between Cdk2 and Cdc7 complexes at the origins [].
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein and/or lipid substrates [, , , , ]. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domainsare often found in clusters, where fingers can have different binding specificities. There are many superfamilies of Znf motifs, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g. some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organisation, epithelial development, cell adhesion, protein folding, chromatin remodelling and zinc sensing, to name but a few []. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. In eukaryotes, initiation of DNA replication requires the assembly of pre-replication complexes (pre-RCs) on chromatin during the G1 phase. In the S phase, pre-RCs are activated by two protein kinases, Cdk2 and Cdc7, which results in the loading of replication factors and the unwinding of replication origins by the MCM helicase complex []. Cdc7 is a serine/threonine kinase that is conserved from yeast to human. It is regulated by its association with a regulatory subunit, the Dbf4 protein. This complex is often referred to as DDK (Dbf4-dependent kinase) [].DBF4 contains an N-terminal BRCT domain and a C-terminal conserved region that could potentially coordinate one zinc atom, the DBF4-type zinc finger. This entry represents the zinc finger, which is important for the interaction with Cdc7 [, ].
The MCM2-7 complex consists of six closely related proteins that are highly conserved throughout the eukaryotic kingdom. In eukaryotes, Mcm2 is a component of the MCM2-7 complex (MCM complex), which consists of six sequence-related AAA + type ATPases/helicases that form a hetero-hexameric ring []. MCM2-7 complex is part of the pre-replication complex (pre-RC). In G1 phase, inactive MCM2-7 complex is loaded onto origins of DNA replication [, , ]. During G1-S phase, MCM2-7 complex is activated to unwind the double stranded DNA and plays an important role in DNA replication forks elongation [].The components of the MCM2-7 complex are: DNA replication licensing factor MCM2, DNA replication licensing factor MCM3, DNA replication licensing factor MCM4, DNA replication licensing factor MCM5, DNA replication licensing factor MCM6, DNA replication licensing factor MCM7, In addition to its role in initiation of DNA replication, Mcm2 is able toinhibit the Mcm4,6,7 helicase. Studies on murine Mcm2 indicate that itsC terminus is required for interaction with MCM4, as well as for inhibitionof the DNA helicase activity of the Mcm4,6,7 complex. The N-terminal region,which contains an H3-binding domain and a region required for nuclear localisation, is required for the phosphorylation by CDC7 kinase.
Small GTPases form an independent superfamily within the larger class of regulatory GTP hydrolases. This superfamily contains proteins that control a vast number of important processes and possess a common, structurally preserved GTP-binding domain [, ]. Sequence comparisons of small G proteins from various species have revealed that they are conserved in primary structures at the level of 30-55% similarity [].Crystallographic analysis of various small G proteins revealed the presence of a 20kDa catalytic domain that is unique for the whole superfamily [, ]. The domain is built of five alpha helices (A1-A5), six β-strands (B1-B6) and five polypeptide loops (G1-G5). A structural comparison of the GTP- and GDP-bound form, allows one to distinguish two functional loop regions: switch I and switch II that surround the gamma-phosphate group of the nucleotide. The G1 loop (also called the P-loop) that connects the B1 strand and the A1 helix is responsible for the binding of the phosphate groups. The G3 loop provides residues for Mg2 and phosphate binding and is located at the N terminus of the A2 helix. The G1 and G3 loops are sequentially similar to Walker A and Walker B boxes that are found in other nucleotide binding motifs. The G2 loop connects the A1 helix and the B2 strand and contains a conserved Thr residue responsible for Mg2 binding. The guanine base is recognised by the G4 and G5 loops. The consensus sequence NKXD of the G4 loop contains Lys and Asp residues directly interacting with the nucleotide. Part of the G5 loop located between B6 and A5 acts as a recognition site for the guanine base [].The small GTPase superfamily can be divided into at least 8 different families, including:Arf small GTPases. GTP-binding proteins involved in protein trafficking by modulating vesicle budding and uncoating within the Golgi apparatus.Ran small GTPases. GTP-binding proteins involved in nucleocytoplasmic transport. Required for the import of proteins into the nucleus and also for RNA export.Rab small GTPases. GTP-binding proteins involved in vesicular traffic.Rho small GTPases. GTP-binding proteins that control cytoskeleton reorganisation.Ras small GTPases. GTP-binding proteins involved in signalling pathways.Sar1 small GTPases. Small GTPase component of the coat protein complex II (COPII) which promotes the formation of transport vesicles from the endoplasmic reticulum (ER).Mitochondrial Rho (Miro). Small GTPase domain found in mitochondrial proteins involved in mitochondrial trafficking.Roc small GTPases domain. Small GTPase domain always found associated with the COR domain.This entry includes Tem1 from budding yeasts and Spg1 from fission yeasts. They are GTPases involved in the regulation of the cell cycle. In Schizosaccharomyces pombe, Spg1 is required for the localisation of Cdc7 (part of the septation initiation network) to the spindle pole body (SPB) []. It is regulated negatively by a GTPase-activating protein (GAP) comprising two subunits - Byr4 and Cdc16. In anaphase B, Spg1 is localised on the new SPB []. In Saccharomyces cerevisiae, Tem1 is associated with the mitotic exit network (MEN). It is involved in termination of M phase of the cell cycle [].
